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Unlike other subclasses of the Retroviridae the Spumavirinae, its prototype member being the so-called human foamy virus (HFV), require the expression of the envelope (Env) glycoprotein for viral particle egress. Both the murine leukemia virus (MuLV) Env and the vesicular stomatitis virus G protein, which efficiently pseudotype other retrovirus capsids, were not able to support export of HFV particles. Analysis of deletion and point mutants of the HFV Env protein revealed that the HFV Env cytoplasmic domain (CyD) is dispensable for HFV particle envelopment, release, and infectivity, whereas deletion of the membrane-spanning-domain (MSD) led to an accumulation of naked capsids in the cytoplasm. Neither alternative membrane association of HFV Env deletion mutants lacking the MSD and CyD via phosphoglycolipid anchor nor domain swapping mutants, with the MSD or CyD of MuLV Env and VSV-G exchanged against the corresponding HFV domains, could restore particle envelopment and the release defect of pseudotypes. However, replacement of the HFV MSD with that of MuLV led to budding of HFV capsids at the intracellular membranes. These virions were of apparently wild-type morphology but were not naturally released into the supernatant and they were noninfectious.

The yeast transcriptional activator Gal4p can bind to sites in nucleosomal DNA in vivo which it is unable to access in vitro. One event which could allow proteins to bind to otherwise inaccessible sites in chromatin in living cells is DNA replication. To determine whether replication is required for Gal4p to bind to nucleosomal sites in yeast, we have used previously characterized chromatin reporters in which Gal4p binding sites are incorporated into nucleosomes. We find that Gal4p is able to perturb nucleosome positioning via nucleosomal binding sites in yeast arrested either in G1, with alpha-factor, or in G2/M, with nocodazole. Similar results were obtained whether Gal4p synthesis was induced from the endogenous promoter by growth in galactose medium or by an artificial, hormone-inducible system. We also examined binding of the Drosophila transcriptional activator Bicoid, which belongs to the homeodomain class of transcription factors. We show that Bicoid, like Gal4p, can bind to nucleosomal sites in SWI+ and swi1Delta yeast and in the absence of replication. Our results indicate that some feature of the intracellular environment other than DNA replication or the SWI-SNF complex permits factor access to nucleosomal sites.

Genetic hemochromatosis (GH) is believed to be a disease restricted to those of European ancestry. In northwestern Europe, >80% of GH patients are homozygous for one mutation, the substitution of tyrosine for cysteine at position 282 (C282Y) in the unprocessed protein. In a proportion of GH patients, two mutations are present, C282Y and H63D. The clinical significance of this second mutation is such that it appears to predispose 1%-2% of compound heterozygotes to expression of the disease. The distribution of the two mutations differ, C282Y being limited to those of northwestern European ancestry and H63D being found at allele frequencies>5%, in Europe, in countries bordering the Mediterranean, in the Middle East, and in the Indian subcontinent. The C282Y mutation occurs on a haplotype that extends </=6 Mb, suggesting that this mutation has arisen during the past 2,000 years. The H63D mutation is older and does not occur on such a large extended haplotype, the haplotype in this case extending </=700 kb. Here we report the finding of the H63D and C282Y mutations on new haplotypes. In Sri Lanka we have found H63D on three new haplotypes and have found C282Y on one new haplotype, demonstrating that these mutations have arisen independently on this island. These results suggest that the HFE gene has been the subject of selection pressure. These selection pressures could be due to infectious diseases, environmental conditions, or other genetic disorders such as anemia.

The testicular isoform of hormone-sensitive lipase (HSLtes) is encoded by a testis-specific exon and 9 exons common to the testis and adipocyte isoforms. In mouse, HSLtes mRNA appeared during spermiogenesis in round spermatids. Two constructs containing 1.4 and 0.5 kilobase pairs (kb) of the human HSLtes gene 5'-flanking region cloned upstream of the chloramphenicol acetyltransferase gene were microinjected into mouse oocytes. Analyses of enzyme activity in male and female transgenic mice showed that 0.5 kb of the HSLtes promoter was sufficient to direct expression only in testis. Cell transfection experiments showed that CREMtau, a testis-specific transcriptional activator, does not transactivate the HSLtes promoter. Using gel retardation assays, four testis-specific binding regions (TSBR) were identified using testis and liver nuclear extracts. The testis-specific protein binding on TSBR4 was selectively competed by a probe containing a SRY/Sox protein DNA recognition site. Sox5 and Sox6 which are expressed in post-meiotic germ cells bound TSBR4. Mutation of the AACAAAG motif in TSBR4 abolished the binding. Moreover, binding of the high mobility group domain of Sox5 induced a bend within TSBR4. Together, our results showed that 0.5 kb of the human HSLtes promoter bind Sox proteins and contain cis-acting elements essential for the testis specificity of HSL.

The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter strain Py2 (Xamo) have been located on a 4.9-kb fragment of chromosomal DNA previously cloned in cosmid pNY2. Sequencing and analysis of the predicted amino acid sequences indicate that the components of Xamo are homologous to those of the aromatic monooxygenases, toluene 2-, 3-, and 4-monooxygenase and benzene monooxygenase, and that the gene order is identical. The genes and predicted polypeptides are aamA, encoding the 497-residue oxygenase alpha-subunit (XamoA); aamB, encoding the 88-residue oxygenase gamma-subunit (XamoB); aamC, encoding the 122-residue ferredoxin (XamoC); aamD, encoding the 101-residue coupling or effector protein (XamoD); aamE, encoding the 341-residue oxygenase beta-subunit (XamoE); and aamF, encoding the 327-residue reductase (XamoF). A sequence with >60% concurrence with the consensus sequence of sigma54 (RpoN)-dependent promoters was identified upstream of the aamA gene. Detailed comparison of XamoA with the oxygenase alpha-subunits from aromatic monooxygenases, phenol hydroxylases, methane monooxygenase, and the alkene monooxygenase from Rhodococcus rhodochrous B276 showed that, despite the overall similarity to the aromatic monooxygenases, XamoA has some distinctive characteristics of the oxygenases which oxidize aliphatic, and particularly alkene, substrates. On the basis of the similarity between Xamo and the aromatic monooxygenases, Xanthobacter strain Py2 was tested and shown to oxidize benzene, toluene, and phenol, while the alkene monooxygenase-negative mutants NZ1 and NZ2 did not. Benzene was oxidized to phenol, which accumulated transiently before being further oxidized. Toluene was oxidized to a mixture of o-, m-, and p-cresols (39.8, 18, and 41.7%, respectively) and a small amount (0.5%) of benzyl alcohol, none of which were further oxidized. In growth studies Xanthobacter strain Py2 was found to grow on phenol and catechol but not on benzene or toluene; growth on phenol required a functional alkene monooxygenase. However, there is no evidence of genes encoding steps in the metabolism of catechol in the vicinity of the aam gene cluster. This suggests that the inducer specificity of the alkene monooxygenase may have evolved to benefit from the naturally broad substrate specificity of this class of monooxygenase and the ability of the host strain to grow on catechol.

Hypercortisolemia is often observed in patients suffering from major depression. As the serotonergic (5-hydroxytryptamine; 5-HT) system plays a major role in the etiology of depression, a loss of endocrine and neurotransmitter system interactions, including corticosterone regulation of 5-HT transporter (5-HTT) and 5-HT receptor expression, may underlie age-related deficits in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis and correlate with an increased incidence of depression with advancing age. In this study, female Fischer 344 rats, ages 3, 13, and 18 months, were bilaterally adrenalectomized and supplemented for 3 weeks with corticosterone (0, 200, or 600 mg; LC, MC, or HC, respectively) containing 21 day sustained-release pellets implanted subcutaneously. Quantitative autoradiography of hippocampal and cortical regions using [3H]citalopram revealed a significant decrease in hippocampal 5-HTT binding in the 3-month HC treatment group compared to age-matched MC and LC groups; this loss was not present in the 13- or 18-month groups. Similarly, quantitative autoradiography using the radiolabeled 5-HT(1A) receptor agonist 8-hydroxy-2-(di-N-propylamino) tetralin demonstrated a significant decline in receptor density in 3- and 13-month MC and HC groups as compared to age-matched LC groups in the hippocampus. These hormone treatments (MC or HC), however, failed to alter hippocampal 5-HT(1A) binding site density in the 18-month groups as compared to the age-matched LC group. The 5-HT(2A) receptor was also evaluated using [3H]ketanserin and showed no age- or corticosterone-related changes in the cortex. Overall, an age-associated deficit in the regulation of the hippocampal serotonergic system by varied corticosterone treatment was revealed in the present study, which may underlie the increased incidence of depression and hypercortisolemia found with advancing age.

Hereditary hemochromatosis (HH) is a common autosomal recessive genetic disorder of iron metabolism. The HFE candidate gene encoding an HLA class I-like protein involved in HH was identified in 1996. Two missense mutations have been described: C282Y, accounting for 80% to 90% of HH chromosomes, and H63D, which is associated with a milder form of the disease representing 40% to 70% of non-C282Y HH chromosomes. We report here on the analysis of C282Y, H63D, and the 193A-->T substitution leading to the S65C missense substitution in a large series of probands and controls. The results confirm that the C282Y substitution was the main mutation involved in hemochromatosis, accounting for 85% of carrier chromosomes, whereas the H63D substitution represented 39% of the HH chromosomes that did not carry the C282Y mutation. In addition, our screening showed that the S65C substitution was significantly enriched in probands with at least one chromosome without an assigned mutation. This substitution accounted for 7.8% of HH chromosomes that were neither C282Y nor H63D. This enrichment of S65C among HH chromosomes suggests that the S65C substitution is associated with the mild form of hemochromatosis.

The success of spermatogenesis is dependent upon closely coordinated interactions between Sertoli cells and germ cells. To identify specific molecules that mediate interactions between somatic cells and germ cells in the rat testis, Sertoli cell-germ cell co-cultures and mRNA differential display were used. Two cDNAs, clone 1 (660 nucleotides) and clone 2 (390 nucleotides) were up-regulated when Sertoli cells were co-cultured with pachytene spermatocytes or round spermatids. Northern blot analyses confirmed the differential display expression patterns. Sequence analyses indicated that clone 1 was similar to a von Ebner's gland protein (87% at the nucleotide level and 80% at the amino acid level) and clone 2 was identical to a region of the Huntington disease protein. The von Ebner's-like protein mRNA was induced after 4 h of co-culture, while the Huntington disease protein required 18 h of co-culture for expression. The von Ebner's-like protein was induced in germ cells by a secreted Sertoli cell factor(s) smaller than 10 kDa that is sensitive to freezing and thawing or boiling. The Huntington disease protein was induced in germ cells by a Sertoli cell secreted factor(s) larger than 10 kDa which survives freezing and thawing, but is inactivated by boiling.

The amylose fraction of starch occurs in double-helical A- and B-amyloses and the single-helical V-amylose. The latter contains a channel-like central cavity that is able to include molecules, "iodine's blue" being the best-known representative. Molecular models of these amylose forms have been deduced by solid state 13C cross-polarization/magic angle spinning NMR and by x-ray fiber and electron diffraction combined with computer-aided modeling. They remain uncertain, however, as no structure at atomic resolution is available. We report here the crystal structure of a hydrated cycloamylose containing 26 glucose residues (cyclomaltohexaicosaose, CA26), which has been determined by real/reciprocal space recycling starting from randomly positioned atoms or from an oriented diglucose fragment. This structure provides conclusive evidence for the structure of V-amylose, as the macrocycle of CA26 is folded into two short left-handed V-amylose helices in antiparallel arrangement and related by twofold rotational pseudosymmetry. In the V-helices, all glucose residues are in syn orientation, forming systematic interglucose O(3)n...O(2)(n+l) and O(6)n...O(2)(n+6)/O(3)(n+6) hydrogen bonds; the central cavities of the V-helices are filled by disordered water molecules. The folding of the CA26 macrocycle is characterized by typical "band-flips" in which diametrically opposed glucose residues are in anti rather than in the common syn orientation, this conformation being stabilized by interglucose three-center hydrogen bonds with O(3)n as donor and O(5)(n+l), O(6)(n+l) as acceptors. The structure of CA26 permitted construction of an idealized V-amylose helix, and the band-flip motif explains why V-amylose crystallizes readily and may be packed tightly in seeds.

Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine [poly(Q)] repeat expansion in the first exon of the huntingtin protein. Previously, we showed that N-terminal huntingtin peptides with poly(Q) tracts in the pathological range (51-122 glutamines), but not with poly(Q) tracts in the normal range (20 and 30 glutamines), form high molecular weight protein aggregates with a fibrillar or ribbon-like morphology, reminiscent of scrapie prion rods and beta-amyloid fibrils in Alzheimer's disease. Here we report that the formation of amyloid-like huntingtin aggregates in vitro not only depends on poly(Q) repeat length but also critically depends on protein concentration and time. Furthermore, the in vitro aggregation of huntingtin can be seeded by preformed fibrils. Together, these results suggest that amyloid fibrillogenesis in Huntington's disease, like in Alzheimer's disease, is a nucleation-dependent polymerization.

We examined a panel of 21 patients diagnosed with compulsive buying for two DNA sequence polymorphisms found in the gene that encodes the serotonin transport (5-HTT). One polymorphism, found in the promoter region of the 5-HTT gene, involves a 44-base pair (bp) deletion, and the other, found in the second intron, is due to variable numbers of a repeat sequence. We also typed a panel of 38 psychiatrically normal controls for both 5-HH markers. When compared to this control panel, no significant differences were seen for either 5-HTT marker among the compulsive buyers.

Forty-eight primary oral squamous cell carcinomas (SCC) were screened for allelic imbalance (AI) at 3p24-26, 3p21, 3p13, 8p21-23, 9p21, 9q22 and within the Rb, p53 and DCC tumour suppressor genes. AI was detected at all TNM stages with stage 4 tumours showing significantly more aberrations than stage 1-3. A factional allelic loss (FAL) score was calculated for all tumours and a high score was associated with development of local recurrence (P = 0.033) and reduced survival (P = 0.0006). AI at one or more loci within the 3p24-26, 3p21, 3p13 and 9p21 regions or within the THRB and DCC genes was associated with reduced survival. The hazard ratios for survival analysis revealed that patients with AI at 3p24-26, 3p13 and 9p21 have an approximately 25 times increase in their mortality rate relative to a patient retaining heterozygosity at these loci. AI at specific pairs of loci, D3S686 and D9S171 and involving at least two of D3S1296, DCC and D9S43, was a better predictor of prognosis than the FAL score or TNM stage. These data suggest that it will be possible to develop a molecular staging system which will be a better predict of outcome than conventional clinicopathological features as the molecular events represent fundamental biological characteristics of each tumour.

<h4>Background/aims</h4>The HFE gene is a crucial candidate gene for hemochromatosis. The aims of this study were to assess the HFE genotypic profile in a large series of unrelated probands diagnosed as having phenotypic hemochromatosis, to characterize the sub-group of patients who were not homozygous for the major C282Y mutation, and to report the iron status of the detected HFE-identical siblings.<h4>Methods</h4>In 217 patients, the phenotypic diagnosis of hemochromatosis was based on strict bioclinical and/or histological criteria, and their genotypic profile (C282Y and H63D mutations) was determined.<h4>Results</h4>1) 209 of the 217 probands were C282Y +/+. In 33 cases, an HFE-identical sibling was identified. Two of them had neither a clinical nor a biochemical phenotypic profile of hemochromatosis in the absence of any external factor which might have attenuated this expression. 2) Eight patients (seven males) were not C282Y +/+. Their genotypic profiles were: (C282Y +/-): six cases (four were H63D +/- and two H63D -/-); (C282Y -/-): two cases (one was H63D +/+, one H63D +/-). Phenotypic expression consisted of six cases of mild liver siderosis (among whom were the four compound heterozygotes and one case of alcoholic cirrhosis) and two severe cases of hepatic iron overload (one with alcoholic cirrhosis). Three HFE-identical siblings were identified, none of them presenting with iron excess.<h4>Conclusions</h4>In our population: 1) The classical phenotypic criteria fitted, in 96.3% of cases, with a homogeneous genotypic entity defined by homozygosity for the C282Y mutation. Incomplete penetrance of the homozygous status was shown by the absence of the hemochromatosis phenotypic profile in 6% of the HFE-identical siblings. 2) A minority (3.7%) were not homozygous for C282Y. These were essentially men with mild iron overload, and might present with distinct iron overload entity(ies) as suggested by the presence in three of an HFE-identical sibling with absence of iron overload.

Chromosome condensation is one of the most critical steps during cell division. However, the structure of condensed mitotic chromosomes is poorly understood. In this paper we describe a new approach based on elasticity measurements for studying the structure of in vitro assembled mitotic chromosomes in Xenopus egg extract. The approach is based on a unique combination of measurements of both longitudinal deformability and bending rigidity of whole chromosomes. By using specially designed micropipettes, the chromosome force-extension curve was determined. Analysis of the curvature fluctuation spectrum allowed for the measurement of chromosome bending ridigity. The relationship between the values of these two parameters is very specific: the measured chromosome flexibility was found to be 2,000 times lower than the flexibility calculated from the experimentally determined Young modulus. This requires the chromosome structure to be formed of one or a few thin rigid elastic axes surrounded by a soft envelope. The properties of these axes are well-described by models developed for the elasticity of titin-like molecules. Additionally, the deformability of in vitro assembled chromosomes was found to be very similar to that of native somatic chromosomes, thus demonstrating the existence of an essentially identical structure.

<h4>Objective</h4>To determine whether the osteoarticular changes associated with genetic hemochromatosis could be explained by metabolic parathyroid hormone (PTH) disorders.<h4>Methods</h4>The study involved 210 patients with liver iron overload syndromes. Osteoarticular changes were numerically scored as the number of damaged joints. PTH 1-84 and 44-68 were assayed.<h4>Results</h4>An increase in serum PTH 44-68 levels was found in one-third of untreated patients who had no calcium or PTH 1-84 abnormalities. Serum PTH 44-68 levels correlated positively with serum ferritin levels. In multivariate analyses, the number of affected joints correlated positively with age, serum PTH 44-68 levels, and serum ferritin levels.<h4>Conclusion</h4>Liver iron overload syndromes, especially genetic hemochromatosis, are associated with elevated circulating levels of PTH fragments containing the 44-68 region, which appears to play a role in osteoarticular changes. This increase seems to be a consequence of iron overload.

Activation of ADP-ribosylation factors (ARFs), approximately 20-kDa guanine nucleotide-binding proteins that play an important role in intracellular vesicular trafficking, depends on guanine nucleotide-exchange proteins (GEPs), which accelerate replacement of bound GDP with GTP. Two major families of ARF GEPs are known: approximately 200-kDa molecules that are inhibited by brefeldin A (BFA), a fungal metabolite that blocks protein secretion and causes apparent disintegration of Golgi structure, and approximately 50-kDa GEPs that are insensitive to BFA. We describe here two human brain cDNAs that encode BFA-inhibited GEPs. One is a approximately 209-kDa protein 99.5% identical in deduced amino acid sequence (1, 849 residues) to a BFA-inhibited ARF GEP (p200) from bovine brain. The other smaller protein, which is approximately 74% identical (1, 785 amino acids), represents a previously unknown gene. We propose that the former, p200, be named BIG1 for (brefeldin A-inhibited GEP1) and the second, which encodes a approximately 202-kDa protein, BIG2. A protein containing sequences found in BIG2 had been purified earlier from bovine brain. Human tissues contained a 7.5-kilobase BIG1 mRNA and a 9.4-kilobase BIG2 transcript. The BIG1 and BIG2 genes were localized, respectively, to chromosomes 8 and 20. BIG2, synthesized as a His6 fusion protein in Sf9 cells, accelerated guanosine 5'-3-O-(thio)triphosphate binding by recombinant ARF1, ARF5, and ARF6. It activated native ARF (mixture of ARF1 and ARF3) more effectively than it did any of the nonmyristoylated recombinant ARFs. BIG2 activity was inhibited by BFA in a concentration-dependent manner but not by B17, a structural analog without effects on Golgi function. Although several clones for approximately 50-kDa BFA-insensitive ARF GEPs are known, these new clones for the approximately 200-kDa BIG1 and BIG2 should facilitate characterization of this rather different family of proteins as well as the elucidation of mechanisms of regulation of BFA-sensitive ARF function in Golgi transport.

Transgenic mice overexpressing cytokines facilitate analysis of the effects of these immunomodulators on indigenous cells of the central nervous system. This study examines morphological aspects of demyelination and permeability changes, in a recently described transgenic model (termed GFAP-IL3). GFAP-IL3 mice develop progressive motor disease at approximately 5 months. Lesions identified after disease onset, showed activation of microglia, astroglial proliferation with phagocytosis of lipids, and immigration of macrophages and mast cells into neural parenchyma. Lymphocytes failed to appear until the later stages of the disease. Later, cerebellar and brain stem white matter contained focal demyelinating lesions with intense macrophage infiltration and a proliferative astrocytosis. Dystrophic axonal changes were noted, in addition to demyelination in heavily infiltrated lesions. Mast cells, variably present in the thalamus and meninges of wild type mice, were greatly increased at these sites in GFAP-IL3 mice. Blood-brain barrier (BBB) defects were documented with leakage of intravenously injected horseradish peroxidase. Mast cell infiltration into the CNS and their degranulation at the site of injury, may represent initial events in a spontaneous process of macrophage mediated demyelination in which glial cells and macrophages are both involved in the phagocytic process.

N-terminal mutations in beta-catenin that inhibit beta-catenin degradation are found in primary tumors and cancer cell lines, and increased beta-catenin/T cell factor (TCF)-activated transcription in these cells has been correlated with cancer formation. However, the role of mutant beta-catenin in cell transformation is poorly understood. Here, we compare the ability of different N-terminal mutations of beta-catenin (DeltaN131, DeltaN90, DeltaGSK) to induce TCF-activated transcription and anchorage-independent growth in Madin-Darby canine kidney epithelial cells. Expression of DeltaN90 or DeltaGSK beta-catenin increased TCF-activated transcription but did not induce significant anchorage-independent cell growth. In contrast, deletion of the alpha-catenin-binding site in DeltaN131 beta-catenin reduced TCF-activated transcription, compared with that induced by DeltaN90 or DeltaGSK beta-catenin, but significantly enhanced anchorage-independent cell growth.

Hereditary haemochromatosis is a common inherited disorder of iron metabolism in Caucasian populations. Two mutations in the HFE gene are strongly associated with hereditary haemochromatosis. One of these mutations (Cys282-->Tyr; C282Y) is found homozygous in 90-95% of subjects with typical hereditary haemochromatosis. A second mutation (His63-->Asp; H63D) has also been identified but is not associated with the same degree of iron overload as with the C282Y mutation. About 20% of subjects who are heterozygous for both mutations (C282Y, H63D-compound heterozygotes) can express typical hereditary haemochromatosis. A large number of patients with early disease are asymptomatic, and prompt diagnosis and treatment can result in normal life expectancy. The diagnosis can readily be confirmed by serum iron studies and genetic testing. For C282Y homozygotes or compound heterozygotes diagnosed under the age of 40 years and with no biochemical or clinical evidence of liver disease, phlebotomy therapy can be initiated without the need for liver biopsy. Liver biopsy should still be considered in all other patients with iron overload. Screening of first degree relatives should now be based on genotype assessment and measurement of serum iron parameters in order to determine phenotypic expression of the disease.

The recent knowledges about the functions of huntingtin and the pathomechanism of Huntington disease are reviewed. Several binding proteins such as HAP1, ubiquitin-conjugating enzyme, HIP1, glyceraldehyde-3-phosphate-dehydrogenase(GAPDH), and cystathionine beta-synthase are identified. One of the functions of huntingtin suggested by those binding proteins is organella transport. In addition huntington binds with WW domain proteins and SH3 domain. The most exciting discovery of Huntington disease pathomechanism is identification of nuclear inclusions in transgenic mouse model of Huntington disease. The discussion about the significance of nuclear inclusion for the cell death was reviewed.

The understanding of iron metabolism at the molecular level has been enormously expanded in recent years by new findings about the functioning of transferrin, the transferrin receptor and ferritin. Other recent developments include the discovery of the hemochromatosis gene HFE, identification of previously unknown proteins involved in iron transport, divalent metal transporter 1 and stimulator of Fe transport, and expanded insights into the regulation and expression of proteins involved in iron metabolism. Interactions among principal participants in iron transport have been uncovered, although the complexity of such interactions is still incompletely understood. Correlated efforts involving techniques and concepts of crystallography, spectroscopy and molecular biology applied to cellular processes have been, and should continue to be, particularly revealing.

A high prevalence of a common mutation in the Hfe gene (C282Y) has recently been reported in patients with the factor V Leiden mutation and a history of thrombosis. The aim of this study was to estimate the relative risk of venous thromboembolism in a large case-control study. 56/481 patients (11.6%) and 57/497 controls (11.5%) were heterozygous for the C282Y allele giving an odds ratio of 1.02 (95%CI 0.69-1.51). 12/81 patients with the factor V Leiden mutation were heterozygous for the C282Y allele compared to 1/13 controls, odds ratio 2.09 (95%CI 0.25-17.6). An analysis of a further group of patients and controls selected for the factor V Leiden mutation did not indicate a higher prevalence of the C282Y allele in symptomatic patients, odds ratio 0.17 (95%CI 0.34-0.81). This study does not support the hypothesis that the C282Y allele is an additional risk factor for venous thrombosis in patients with the factor V Leiden mutation.

The study of misplaced endometrial cells, which abnormally implant and grow outside the uterine cavity, is of considerable interest for the understanding of the pathophysiology of endometriosis. However, endometriotic cells, particularly epithelial cells, required for primary cell culture are not easily available. We report here the characterization of an endometriotic cell line immortalized after infection of primary endometriotic cell cultures with simian virus 40. Transformed cells express T-antigen, and blot hybridization analysis showed that the viral genome is present as an episome. Cytogenetic analysis revealed a polyploid karyotype with numerical and structural rearrangements involving mainly the same chromosomes (6, 10, 11, 15, and 17). The cell line has been maintained in culture for over 80 passages and was still proliferating without any noticeable change in the biological properties investigated. Transformed endometriotic cells expressed both progesterone and estradiol receptors and were stimulated by these ovarian hormones to secrete monocyte chemotactic protein-1, a factor that may play an important role in the recruitment and activation of peritoneal macrophages. In addition, this response was enhanced in interleukin-1-treated cells. Taken together, these findings support the view that this cell line may be an interesting tool for the study of the pathophysiology of endometriosis.

It is well known that alcoholic liver disease is associated with iron overload. To study the role of hemochromatosis gene mutations on the pathogenesis of alcoholic liver disease (ALD), we have analyzed C282Y and H63D mutations on the chromosomes obtained from 95 Japanese alcoholics. Patients were divided in two groups [i.e., 64 alcoholic patients with liver damage (group I) and 31 alcoholics without liver damage (group II)]. In group I, biochemical examinations showed that serum levels of iron and ferritin were significantly high, and unsaturated iron binding capacity levels were low, compared with those of group II. An analysis by means of allele-specific polymerase chain reaction demonstrated that C282Y mutation was not observed in both groups I and II. H63D mutation was observed in only two heterozygotes of group I and in one heterozygote of group II. Results could not indicate the relationship between ALD and these mutations. We speculate that other causes of iron overload may exist in ALD with iron overload.

<h4>Background</h4>The aim of our study was to evaluate the prevalence of Cys282Tyr mutation in patients with genetic haemochromatosis (GH) in Cantabria.<h4>Patients and methods</h4>The HFE Cys282Tyr mutation was determined in a cohort of 60 patients with GH and 213 controls.<h4>Results</h4>The frequency of the Cys282Tyr mutation in control individuals was 4.4%. Sixty-seven percent of patients with GH were homozygous for the Cys282Tyr mutation. Twenty-seven percent of patients were normal at Cys282Tyr loci.<h4>Conclusions</h4>The prevalence of the Cys282Tyr mutation in patients with GH in Cantabria, Spain, seems to be lower than in North America and in North Europe patients.

We report the case of a 14-year-old girl who originally presented at the age of eight with a history of bloody stools, abdominal pain and weight loss. Initial iron studies showed raised serum iron and transferrin saturation but low ferritin and were interpreted as consistent with iron deficiency under treatment. As she had not taken any supplemental iron she later underwent genetic testing for the Cys282Tyr and His63Asp mutations of the HFE gene. On the basis of these results, she was diagnosed as having hereditary haemochromatosis (HH). This case highlights that a low serum ferritin does not exclude the diagnosis of HH and that the availability of genetic testing can now enable probands and affected family members to be identified.

Genetic and environmental factors have been implicated in the development of Alzheimer's disease (AD), the most common form of dementia in the elderly. Mutations in 3 genes mapped on chromosomes 21, 14 and 1 are related to the rare early onset forms of AD while the epsilon 4 allele of the apolipoprotein E (APOE) gene (on chromosome 19) is the major susceptibility locus for the most common late onset AD (LOAD). Serotonin (5-hydroxytryptamine or 5-HT) is a key neurotransmitter implicated in the control of mood, sleep, appetite and a variety of traits and behaviors. Recently, a polymorphism in the transcriptional control region upstream of the 5-HT transporter (5-HTT) gene has been studied in several psychiatric diseases and personality traits. It has been demonstrated that the short variant(s) of this 5-HTT gene-linked polymorphic region (5-HTTLPR) is associated with a different transcriptional efficiency of the 5-HTT gene promoter resulting in decreased 5-HTT expression and 5-HT uptake in lymphocytes. An increased frequency of this 5-HTTLPR short variant polymorphism in LOAD was recently reported. In addition, another common polymorphic variation in the 5-HT2A and 5-HT2C serotonin receptor genes previously analyzed in schizophrenic patients was associated with auditory and visual hallucinations in AD. These observations suggest that the involvement of the serotonin pathway might provide an explanation for some aspects of the affective symptoms commonly observed in AD patients. In summary, research on genetic polymorphisms related to AD and involved in receptors, transporter proteins and the enzymatic machinery of serotonin might enhance our understanding of this devastating neurodegenerative disorder.

The Deleted in Colorectal Cancer gene (DCC) encodes a cell surface receptor that belongs to the Ig superfamily. Inactivation of the DCC gene has been implicated in human tumor progression. However, little is known about the biological function of the DCC protein. In the present study, we demonstrated that expression of DCC activated caspase-3 and programmed cell death, or induced G2/M cell cycle arrest in tumor cells. In some cell lines, apoptosis was evident within 24 h of DCC expression. Timing of the appearance of apoptotic cells coincided with that of the cleavage of poly (ADP-ribose) polymerase, a substrate of caspase-3. Expression of the apoptosis inhibitory gene Bcl-2 was not able to abrogate the DCC-induced apoptosis. In the G2/M cycle arrest cells, cdk1 activity was inhibited. Our results suggest that the DCC protein may transduce signals resulting in activation of caspases or inhibition of Cdk1. These data provide a possible mechanism by which DCC suppresses tumorigenesis.

On 18q, frequently deleted in late stage colorectal cancers, a gene, Deleted in Colon Cancer (DCC), has been identified and postulated to play a role as a tumor suppressor gene. DCC is retained in the majority of mucinous tumors, which produce high levels of mucins, and seems to be preferentially expressed in intestinal goblet cells. To investigate whether DCC is related to mucin expression and can modulate the transformed phenotype, we introduced a full-length DCC cDNA into HT29 cells, which can be induced in vitro to express MUC2, the gene that encodes the major colonic mucin. Expression of DCC did not modulate constitutive or induced expression of MUC2, nor did DCC induce a mature goblet cell phenotype. However, HT29 clones expressing high and low levels of DCC protein showed a significant decrease in cell proliferation and tumorigenicity. Furthermore, increased shedding and an elevated rate of spontaneous apoptosis were associated with higher levels of expression of DCC. In summary, while restoration of DCC expression in a human colon carcinoma cell line did not influence expression of differentiation markers, DCC expression did affect the growth and tumorigenic properties of the cells suggesting that DCC can modulate the malignant phenotype of colon cancer.

Genetic haemochromatosis is a HLA-linked disease characterized by a high and inappropriate gastrointestinal iron absorption; the excess iron is stored in parenchymal cells, provoking the failure of the involved organs. The common target organs of genetic haemochromatosis are liver, heart, pancreas, pituitary, joints and skin. The disease is inherited in an autosomal recessive manner with predilection for male sex and penetration conditioned by age, sex and food habits. The first clinical manifestation of genetic haemochromatosis, whose onset is typically between ages 40 and 60, is represented by arthropathy in 45% of the cases; the articular features are unfortunately often misdiagnosed and it is known that the diagnosis delay heavily compromises the outcome; vice-versa the early identification of the disease, and the consequent suitable treatment give back a normal life expectancy to these patients. The articular features of genetic haemochromatosis are of two types: 1) progressive degenerative arthropathy, characterized by pain without inflammatory signs, morning stiffness and functional impairment involving hands, wrists, shoulders, hips, knees and feet; 2) chondrocalcinosis with its typical proteiform clinical manifestations. The aim of this report is to underline that the patients with premature osteoarthritis or unexplained chondrocalcinosis must be screened for genetic haemochromatosis in order to formulate the correct diagnosis before the development of severe internal organ involvement.

To determine whether there is an association of polymorphic variants of the serotonin transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) and autistic spectrum disorders, we analyzed the 5-HTTLPR genotypes of 72 autistic subjects, 11 fragile X syndrome patients with autistic behavior, 43 normal subjects, and 49 fragile X syndrome non-autistic subjects. The distribution frequency of 5-HTTLPR long allele (L) and the short allele (S) variants showed no differences between subjects. Our findings do not support the hypothesis that polymorphic 5-HTTLPR variants are a susceptibility factor for autistic disorders.

Selection of a pathway of differentiation in multipotent progenitor cells may depend on the amount of histone H1 or H1 zero relative to the core histones. With low levels of these linker histones, it is proposed that an evolutionarily more ancient cell differentiation occurs. Greater repetition of AT-rich regulatory motifs allows more frequent, hence earlier, transcription of genes, accounting for this type of cell differentiation. It is further proposed that a decrease of total cell protein accumulation leads to an increase of histone H1 or H1 zero relative to the core histones. It is suggested that these linker histones preferentially bind the more AT-rich regulatory sequences, thereby restricting the phylogenetically more ancient differentiation potency. This allows differentiation of an evolutionarily younger cell type.

To clarify possible roles of DCC expression in tumour differentiation and cell kinetics, we immunohistochemically investigated 80 uterine cervical adenocarcinomas (C-ACs), including 31 mucinous (M) and 31 endometrioid (E) lesions, and 18 adenocarcinomas in situ (AIS), along with 39 normal cervical samples. The results were compared with findings for p21(WAF1/Cip1) and p27Kip1 expression, apoptosis, cell proliferation and human papillomavirus (HPV) infection. Nine C-AC cases were also examined using a combination of the reverse transcription-polymerase chain reaction and Southern blot hybridization, as well as Western blot assays. Significantly decreased DCC scores were observed in E-ACs but not M-ACs, as compared to normal cervical glandular epithelia and AIS. Average p21(WAF1/Cip1) and p27Kip1 scores were significantly higher in E-ACs than M-ACs, in line with high apoptotic, mitotic and Ki-67 labelling indices. A concordance of the results for DCC and p21(WAF1/Cip1) expression between mRNA- and protein-based assays was also noted. Change of DCC expression, however, was not related to any of the cell kinetic markers or clinicopathological features in ACs of either type. There was also no association with the HPV status, although infection was significantly linked with high values for cell kinetics. These results suggest that DCC expression in C-ACs is closely associated with mucinous differentiation.

Retinoids exhibit multiple functions through interaction with nuclear retinoid receptors and have growth-suppressive activity on gastric cancer cells. To better understand the roles of nuclear retinoid receptors during gastric carcinogenesis, we have used in situ hybridization to investigate expression of retinoic acid receptors (RARs) and retinoid x receptors (RXRs) in premalignant and malignant formalin-fixed paraffin-embedded gastric tissues. Histological sections of eight normal, 17 distal normal and nine gastric cancer tissues were hybridized with non-radioactive RNA probes for subtypes of RAR and RXR. Expression of RAR alpha, RAR beta, RAR gamma, RXR alpha and RXR beta was found in most cell types in gastric mucosa tissues from normal individuals as well as in distal normal tissues from cancer patients. Expression of RAR alpha and RAR beta were found in three and seven cancer tissues, respectively, and levels of RXR alpha mRNA were significantly decreased in poorly differentiated cancer tissues. Among the five investigated nuclear retinoid receptors, only expression of RAR alpha mRNA was significantly decreased in intestinal metaplasia, dysplasia and cancer tissues when compared to adjacent normal tissues. In conclusion, normal gastric mucosa expressed both RARs and RXRs, which supports the physiological role of retinoic acid on normal gastric mucosa. The decrease in RAR alpha expression in premalignant and malignant gastric tissues suggests a significant role of RAR alpha during gastric carcinogenesis.

Mucormycosis is a rare and invasive mycotic opportunistic infection, occurring mostly in predisposed patients, mainly diabetics and immunocompromised individuals. The evolution of this fungal infection is frequently fatal unless aggressive treatment is started, or predisposing factors are handled. Our first patient was a known diabetic who had ketoacidotic coma at admission, complicated with pulmonary mucormycosis, and needed surgical resection followed by antimycotic therapy. The second patient did not survive his severe aplastic anemia (with neutropenia) and hemochromatosis (treated with desferrioxamine), complicated with a systemic Rhizopus infection, despite treatment with amphotericin B and granulocyte-colony-stimulating factors.

<h4>Objective of study</h4>Assess interrelations between parameters of haemostasis, fibrinolysis, acute stage proteins and cytoadhesive molecules in gestational diabetes (GDM), which in susceptible women is actively induced by insulin resistance.<h4>Methods</h4>In 33 pregnant women where based on the glucose tolerance test (75 g glucose) GDM was revealed, repeatedly fibrinogen values were assessed (FBG 4.38 +/- 0.98 g/l), the antithrombin III activity (ATIII% 103.83 +/- 14.9), t-PA Ag(4.82 +/- 3.35 ng/ml), PAI-1 Ag (158.04 +/- 57.7 ng/ml), D-dimer (234.99 +/- 455.36 ng/ml), plasma proteins-orosomucoid (ORM 0.506 +/- 0.14 g/l), C reactive protein (1.34 +/- 0.96 g/l), transferrin (3.77 +/- 0.79 g/l), prealbumin (1.86 +/- 0.83 g/l) and alpha-2-macroglobulin (A2M 2.01 +/- 0.62 g/l) and cytadhesive molecules sICAM-1 (230.8 +/- 57.76 ng/ml) and sE-selectin (35.50 +/- 16.55 ng/ml). These findings were compared with parameters of the glucose and lipid metabolism and some anthropometric data (BMI, weight increments during pregnancy and birth weight of the infant).<h4>Results</h4>After statistical evaluation in GDM positive correlations of FBG and BMI were found (r = 0.871, p = 0.007) and also with the weight increment during pregnancy (r = 0.934, p = 0.0007). Hyperfibrinogenaemia is however probably compensated in stabilized cases of GDM by a higher production of t-PA (FBG:t-PA Ag, r = 0.782, p = 0.003). In GDM a marginal positive correlation between the value of the "atheroindex" and the infant's length was found r = 0.744 p = 0.0547). sE-selectin levels in GDM correlated inversely with ATIII activity (r = -0.905, p = 0.0028).<h4>Conclusion</h4>The higher body weight of pregnant women with GDM is probably associated with a higher risk of thrombophilia (elevated fibrinogen). If in pregnant women infection develops with release of inflammatory cytokines, inducing on the endothelium expression of E-selectin, deterioration of thrombophilia (due to a decline of ATIII) activity must be foreseen.

Targeted Genetics is developing, tgDCC-E1A, a E1A tumor suppressor gene therapy formulated in a non-viral, lipid-based delivery system as a potential treatment for solid tumors which overexpress the her-2/neu oncogene [221611]. Fournier is the European development partner [244180]. Preclinical studies of E1A in mouse models for human breast and ovarian cancer demonstrated inhibition of expression of the her-2/neu oncogene, a significant reduction in tumors and increased survival rate in the treated animals [244180]. Results of a phase I study of intratumoral liposomal-E1A gene therapy in patients with recurrent/refractory breast cancer and head and neck cancer were presented at the 1998 American Society of Clinical Oncology (ASCO) meeting. Results demonstrated that intratumoral delivery of the E1A gene caused a subsequent downregulation of HER-2/neu expression and tumor response. The phase I dose-escalation study treated nine patients with recurrent breast cancer and nine patients with head and neck cancer. In 16 patients evaluable for response, nine had stable disease, five had progressive disease and two had minor responses despite tumor progression at other sites. In one of six patients who had repeat biopsies of treated tumors, no pathological evidence of tumor was found. In four of seven patients evaluated to date (May 1998), evidence of downregulation of HER-2/neu was reported [287387,290118]. The first phase I trial in patients with breast and ovarian cancer was completed by the end of 1997. The second phase I trial, enrolling 12 to 24 patients with solid tumors, was initiated in April 1997. Its aim was to determine dosing and safety of tgDCC-E1A and evaluate levels of gene transfer and tumor response. Patients were administered tgDCC-E1A by intratumoral injections. The company completed this trial by the end of 1997. In October 1998, Targeted Genetics initiated a multicenter, open-label phase II clinical trial of tgDCC-E1A in patients with recurrent head and neck squamous cell carcinoma who have exhibited disease progression despite standard therapies. The study to be conducted at six medical institutions in the US will enrole up to 60 patients with an interim analysis scheduled to be performed after the first 20 evaluable patients have been enrolled. Patients will be treated with ten direct intratumoral injections of the E1A gene over a period of 8 weeks, with follow-up performed at week 12. Patients who demonstrate a response will be eligible for continued treatment and will be monitored for 1 year [300424]. Targeted Genetics has issued three patents covering methods and compositions of use of the E1A and LTgenes (qv) to treat a variety of cancers. US-05651964 covers the use of the E1A gene, US-05643567 and US-05641484 cover the use of either the E1A gene or the LT gene as cancer therapies. These three patents provide the company with broad patent protection for tgDCC-E1A and all future products based on the use of the E1A or LT gene as tumor suppressors [259808].

Genetic recombination is the creation of new gene combinations in a cell or gamete, which differ from those of progenitor cells or parental gametes. In eukaryotes, recombination may occur at mitosis or meiosis. Mitotic recombination plays an indispensable role in DNA repair, which presumably directed its early evolution; the multiplicity of recombination genes and pathways may be best understood in this context, although they have acquired important additional functions in generating diversity, both somatically (increasing the immune repertoire) and in germ line (facilitating evolution). Chromosomal homologous recombination and HsRad51 recombinase expression are increased in both immortal and preimmortal transformed cells, and may favor the occurrence of multiple oncogenic mutations. Tumorigenesis in vivo is frequently associated with karyotypic instability, locus-specific gene rearrangements, and loss of heterozygosity at tumor suppressor loci - all of which can be recombinationally mediated. Genetic defects which increase the rate of somatic mutation (several of which feature elevated recombination) are associated with early incidence and high risk for a variety of cancers. Moreover, carcinogenic agents appear to quite consistently stimulate homologous recombination. If cells with high recombination arise, either spontaneously or in response to "recombinogens," and predispose to the development of cancer, what selective advantage could favor these cells prior to the occurrence of growth-promoting mutations? We propose that the augmentation of telomere-telomere recombination may provide just such an advantage, to hyper-recombinant cells within a population of telomerase-negative cells nearing their replicative (Hayflick) limit, by extending telomeres in some progeny cells and thus allowing their continued proliferation.