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Mouse embryos nullizygous for a targeted disruption of the Huntington's disease gene homologue (Hdh), which encodes a protein (huntingtin) of unknown biochemical function, become developmentally retarded and disorganized, and die early in development. Using chimeric analysis, we demonstrate that extensively chimeric embryos derived by injection of Hdh null ES cells into wild-type host blastocysts are rescued from lethality. In contrast, when wild-type ES cells are injected into Hdh null blastocysts, the chimeric embryos are morphologically indistinguishable from Hdh null mutants derived from natural matings, and die shortly after gastrulation. Therefore, the primary defect in the absence of huntingtin lies in extraembryonic tissues, whereas the epiblast and its derivatives are affected secondarily. It is likely that the mutation results in impairment of the nutritive functions of the visceral endoderm, which otherwise appears to differentiate normally, as evidenced by the expression of several specific marker genes. Consistent with preliminary histochemical analysis indicating that at least the transport of ferric ions is defective in Hdh mutants and in conjunction with the known localization of huntingtin in the membranes of vesicles associated with microtubules, we hypothesize that this protein is involved in the intracellular trafficking of nutrients in early embryos.

The present study is an analysis of the frequencies of HFE mutations in patients with different forms of iron overload compared with the frequencies found in healthy subjects from the same region. The frequencies of HLA-A and -B antigens and HLA haplotypes were also analyzed in the same subjects. The study population included: 71 healthy individuals; 39 genetically and clinically well-characterized patients with genetic hemochromatosis (HH); and 25 patients with non-classical forms of iron overload (NCH), excluding secondary hemochromatosis. All subjects were HLA-typed and HFE-genotyped by the oligonucleotide ligation assay (OLA). The gene frequencies found for the C282Y and H63D mutations of HFE were respectively: 0.03 and 0.23 in healthy individuals, 0.86 and 0.04 in HH patients, and 0.08 and 0.48 in NCH patients. An expected significant association between HH and HLA-A3 was observed, which was found to be in linkage disequilibrium with the C282Y mutation. A new association was seen, however, between HLA-A29 and NCH, in linkage disequilibrium with the H63D mutation. Again as expected, the HLA-B antigen B7 was associated with HH in linkage disequilibrium with HLA-A3. In addition, the HLA-B antigen B44 was found to be associated with NCH but not in linkage disequilibrium with either A29 or the H63D mutation. In conclusion, a new association of the HFE H63D mutation with forms of hemochromatosis other than HH and a new association between the HLA phenotype A29 and the HFE H63D mutation were found in the same patients. These findings reinforce evidence for the involvement of the major histocompatibility class I in iron metabolism, supporting the notion of a physiological role for the immunological system in the regulation of iron load.

In genetic hemochromatosis (GH), iron overload affects mainly parenchymal cells, whereas little iron is found in reticuloendothelial (RE) cells. We previously found that RE cells from GH patients had an inappropriately high activity of iron regulatory protein (IRP), the key regulator of intracellular iron homeostasis. Elevated IRP should reflect a reduction of the iron pool, possibly because of a failure to retain iron. A defect in iron handling by RE cells that results in a lack of feedback regulation of intestinal absorption might be the basic abnormality in GH. To further investigate the capacity of iron retention in RE cells of GH patients, we used inflammation as a model system as it is characterized by a block of iron release from macrophages. We analyzed the iron status of RE cells by assaying IRP activity and ferritin content after 4, 8, and 24 hours of incubation with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). RNA-bandshift assays showed that in monocytes and macrophages from 16 control subjects, IRP activity was transiently elevated 4 hours after treatment with LPS and IFN-gamma but remarkably downregulated thereafter. Treatment with NO donors produced the same effects whereas an inducible Nitric Oxide Synthase (iNOS) inhibitor prevented them, which suggests that the NO pathway was involved. Decreased IRP activity was also found in monocytes from eight patients with inflammation. Interestingly, no late decrease of IRP activity was detected in cytokine-treated RE cells from 12 GH patients. Ferritin content was increased 24 hours after treatment in monocytes from normal subjects but not in monocytes from GH patients. The lack of downregulation of IRP activity under inflammatory conditions seems to confirm that the control of iron release from RE cells is defective in GH.

It has been suggested that high iron stores enhance colon carcinogenesis. The effect of high dietary iron (Fe) on indices of iron, copper (Cu) and manganese (Mn) status, lipid peroxidation using the thiobarbituric acid reactive substances assay, superoxide dismutase, glutathione peroxidase, glutathione transferase and ceruloplasmin activities, cell proliferation and development of preneoplastic lesions known as aberrant crypt foci (ACF) in rat colon was examined using a 3 x 2 factorial design. Male weanling Sprague-Dawley rats were fed adequate (AFe; 45 mg Fe/kg diet), moderately high (MHFe; 225 mg Fe/kg diet) and high (HFe; 450 mg Fe/kg diet) dietary Fe for 2.5 wk, then treated with azoxymethane (AOM; 2 injections, 1 wk apart; total dose 30 mg/kg body weight) or saline (n = 14-15 per group). Dietary treatment continued for another 6 wk after the second AOM dose. At the time of AOM injection, colon Fe concentrations were one- and threefold higher for MHFe and HFe rats, respectively, than for AFe rats. It was proposed that high dietary Fe would adversely affect Cu and Mn status, resulting in impaired antioxidant enzyme activity. However, neither indices of Cu and Mn status nor colonic mucosal antioxidant enzyme activities were affected by dietary Fe except for plasma ceruloplasmin activity, which was slightly lower in rats fed high iron diets than in rats fed adequate iron diets (P < 0.01). Dietary Fe had no significant effect on colonic mucosal lipid peroxidation, cell proliferation or ACF development. In conclusion, our findings suggest that dietary Fe concentrations that are approximately 5 and 10 times adequate do not enhance oxidative stress, cell proliferation and ACF development in the colon of rats.

We previously reported the development of an in vitro adeno-associated virus (AAV) DNA replication system. The system required one of the p5 Rep proteins encoded by AAV (either Rep78 or Rep68) and a crude adenovirus (Ad)-infected HeLa cell cytoplasmic extract to catalyze origin of replication-dependent AAV DNA replication. However, in addition to fully permissive DNA replication, which occurs in the presence of Ad, AAV is also capable of partially permissive DNA replication in the absence of the helper virus in cells that have been treated with genotoxic agents. Limited DNA replication also occurs in the absence of Ad during the process of establishing a latent infection. In an attempt to isolate uninfected extracts that would support AAV DNA replication, we discovered that HeLa cell extracts grown to high density can occasionally display as much in vitro replication activity as Ad-infected extracts. This finding confirmed previous genetic analyses which suggested that no Ad-encoded proteins were absolutely essential for AAV DNA replication and that the uninfected extracts should be useful for studying the differences between helper-dependent and helper-independent AAV DNA replication. Using specific chemical inhibitors and monoclonal antibodies, as well as the fractionation of uninfected HeLa extracts, we identified several of the cellular enzymes involved in AAV DNA replication. They were the single-stranded DNA binding protein, replication protein A (RFA), the 3' primer binding complex, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA). Consistent with the current model for AAV DNA replication, which requires only leading-strand DNA synthesis, we found no requirement for DNA polymerase alpha-primase. AAV DNA replication could be reconstituted with purified Rep78, RPA, RFC, and PCNA and a phosphocellulose chromatography fraction (IIA) that contained DNA polymerase activity. As both RFC and PCNA are known to be accessory proteins for polymerase delta and epsilon, we attempted to reconstitute AAV DNA replication by substituting either purified polymerase delta or polymerase epsilon for fraction IIA. These attempts were unsuccessful and suggested that some novel cellular protein or modification was required for AAV DNA replication that had not been previously identified. Finally, we also further characterized the in vitro DNA replication assay and demonstrated by two-dimensional (2D) gel electrophoresis that all of the intermediates commonly seen in vivo are generated in the in vitro system. The only difference was an accumulation of single-stranded DNA in vivo that was not seen in vitro. The 2D data also suggested that although both Rep78 and Rep68 can generate dimeric intermediates in vitro, Rep68 is more efficient in processing dimers to monomer duplex DNA. Regardless of the Rep that was used in vitro, we found evidence of an interaction between the elongation complex and the terminal repeats. Nicking at the terminal repeats of a replicating molecule appeared to be inhibited until after elongation was complete.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease associated with a CAG trinucleotide repeat expansion in a large gene on chromosome 4. The gene encodes the protein huntingtin with a polyglutamine tract encoded by the CAG repeat at the N-terminus. The number of CAG repeats in HD are significantly increased (36 to 120+) compared with the normal population (8-39). The pathological mechanism associated with the expanded CAG repeat in HD is not clear but there is evidence that polyglutamine is directly neurotoxic. We have immunolocalized huntingtin with an in-house, well-characterised, polyclonal antibody in HD, Alzheimer's disease (AD), and Picks disease (PiD) brains. Control brain tissue sections were from head injured and cerebral ischaemia cases. In HD, huntingtin was immunopositive in the surviving but damaged neurons and reactive astrocytes of the caudate and putamen. However, in AD and PiD the immunostaining was largely restricted to the characteristic intracellular inclusion bodies associated with the disease process in each case. In AD, huntingtin was localized only in the intracellular neurofibrillary tangles and dystrophic neurites within the neuritic amyloid plaques but not with the amyloid. In PiD, strongly positive huntingtin immunostaining was present within cytoplasmic Pick bodies. Our findings suggest huntingtin selectively accumulates in association with abnormal intracytoplasmic and cytoskeletal filaments of neurons and glia in neurodegenerative diseases such as HD, AD, and PiD. Cells in the CNS appear sensitive to damage by the aggregated, toxic levels of huntingtin and evidence of its interaction with neurofilaments could provide information about its potential role in the aetiology of HD.

Recently, a novel gene of the major histocompatibility complex (MHC) class I family, HFE (HLA-H), has been found to be mutated in a large proportion of hereditary hemochromatosis (HH) patients. Further support for a causative role of HFE in this disease comes from the observation that beta2-microglobulin knockout (beta2m-/-) mice, that fail to express MHC class I products, develop iron overload. We have now used this animal model of HH to examine the capacity to adapt iron absorption in response to altered iron metabolism in the absence of beta2m-dependent molecule(s). Mucosal uptake, mucosal transfer and retention of iron were measured in control and beta2m-/- mice with altered iron metabolism. Mucosal uptake of Fe(III), but not of Fe(II), by the mutant mice was significantly higher when compared with B6 control mice. Mucosal transfer in the beta2m-/- mice was higher, independent of the iron form tested. No significant differences were found in iron absorption between control and beta2m-/- mice when anemia was induced either by repetitive bleeding or by hemolysis through phenylhydrazine treatment. However, iron absorption in mice made anemic by dietary deprivation of iron was significantly higher in the mutant mice. Furthermore, the beta2m-/- mice manifested an impaired capacity to downmodulate iron absorption when dietary or parenterally iron-loaded. The expression of the defect in iron absorption in the beta2m-/- mice is quantitative, with iron absorption being excessively high for the size of body iron stores. The higher iron absorption capacity in the beta2m-/- mice may involve the initial step of ferric mucosal uptake and the subsequent step of mucosal transfer of iron to the plasma.

Proteins that can be shown to strongly bind in vitro to the four-way (Holliday) junction DNA include not only the obvious candidates such as enzymes involved in recombination, but also a remarkably diverse group of seemingly unrelated proteins. These include the HMG1 box proteins, members of the HMGI-Y family, winged helix proteins (including linker histones), the SWI/SNF complex, and some totally unrelated prokaryotic proteins. What these proteins seem to share is a propensity to bind to bent DNA, to bend DNA upon binding, and/or to preferentially interact with DNA crossings. Thus, they appear to be, in the main, architectural proteins, although some (like the SWI/SNF complex) have very specific functional roles as well. Perhaps because they bind to or promote the formation of particular DNA structures, the four-way junction binding proteins are frequently interchangeable in cellular function. Furthermore, since a given kind of structure can be recognized by many different protein motifs, it is not surprising that apparently unrelated proteins can fall into such a single functional class.

The neurodegenerative diseases Huntington disease, dentatorubropallidoluysian atrophy, spinocerebellar atrophy type 3, and spinal bulbar muscular atrophy are caused by expansion of a polyglutamine tract within their respective gene products. There is increasing evidence that generation of truncated proteins containing an expanded polyglutamine tract may be a key step in the pathogenesis of these disorders. We now report that, similar to huntingtin, atrophin-1, ataxin-3, and the androgen receptor are cleaved in apoptotic extracts. Furthermore, each of these proteins is cleaved by one or more purified caspases, cysteine proteases involved in apoptotic death. The CAG length does not modulate susceptibility to cleavage of any of the full-length proteins. Our results suggest that by generation of truncated polyglutamine-containing proteins, caspase cleavage may represent a common step in the pathogenesis of each of these neurodegenerative diseases.

A previously undescribed Helicobacter sp. was recovered from a cat with severe diarrhea. Based upon the absence of any other identifiable cause of diarrhea, this helicobacter may be involved in the development of the disease signs. The organism could not be cultured but was described on the basis of 16S rRNA gene sequence analysis and morphology and appeared to be a new species, with Helicobacter canis being the most genetically similar species. The presence of a diarrhea-inducing helicobacter in a companion animal may pose a risk of zoonosis.

HFE is an MHC-related protein that is mutated in the iron-overload disease hereditary hemochromatosis. HFE binds to transferrin receptor (TfR) and reduces its affinity for iron-loaded transferrin, implicating HFE in iron metabolism. The 2.6 A crystal structure of HFE reveals the locations of hemochromatosis mutations and a patch of histidines that could be involved in pH-dependent interactions. We also demonstrate that soluble TfR and HFE bind tightly at the basic pH of the cell surface, but not at the acidic pH of intracellular vesicles. TfR:HFE stoichiometry (2:1) differs from TfR:transferrin stoichiometry (2:2), implying a different mode of binding for HFE and transferrin to TfR, consistent with our demonstration that HFE, transferrin, and TfR form a ternary complex.

The sodium-dependent, high affinity serotonin [5-hydroxytryptamine (5-HT)] transporter (5-HTT) provides the primary mechanism for inactivation of 5-HT after its release into the synaptic cleft. To further evaluate the function of the 5-HTT, the murine gene was disrupted by homologous recombination. Despite evidence that excess extracellular 5-HT during embryonic development, including that produced by drugs that inhibit the 5-HTT, may lead to severe craniofacial and cardiac malformations, no obvious developmental phenotype was observed in the 5-HTT-/- mice. High affinity [3H]5-HT uptake was completely absent in 5-HTT-/- mice, confirming a physiologically effective knockout of the 5-HTT gene. 5-HTT binding sites labeled with [125I] 3 beta-(4'-iodophenyl)tropan-2 beta-carboxylic acid methyl ester were reduced in a gene dose-dependent manner, with no demonstrable binding in 5-HTT-/- mutants. In adult 5-HTT-/- mice, marked reductions (60-80%) in 5-HT concentrations were measured in several brain regions. While (+)-amphetamine-induced hyperactivity did not differ across genotypes, the locomotor enhancing effects of (+)-3, 4-methylenedioxymethamphetamine, a substituted amphetamine that releases 5-HT via a transporter-dependent mechanism, was completely absent in 5-HTT-/- mutants. Together, these data suggest that the presence of a functional 5-HTT is essential for brain 5-HT homeostasis and for 3,4-methylenedioxymethamphetamine-induced hyperactivity.

The NM23-H1 gene product has been recently identified as a potential metastasis suppressor. Studies on breast carcinomas have shown an inverse correlation between NM23-H1 status and stage of carcinogenesis and overall survival. However, in colorectal cancer, conflicting data have been reported. This study aimed to investigate whether NM23-H1 immunostaining is correlated with tumour stage, overall survival, disease recurrence, tumour differentiation, age and sex in colorectal carcinomas for the Singapore population using chi-square analysis. The staining was performed on 141 paraffin-embedded surgical specimens collected between 1991 and 1992 using a monoclonal anti-NM23-H1 antibody. Follow-up of patients was until time of death or for 5 years. There was a very significant inverse association between tumour staging and NM23-H1 status (P = 0.0004). However, NM23-H1 expression was not significantly correlated to overall 5-year survival, disease recurrence, tumour differentiation, age or sex. Thus, although NM23-H1 may be involved in suppressing metastasis, NM23-H1 immunohistochemistry has no prognostic value in colorectal cancer. This is the first report of a significant inverse association of NM23-H1 status with tumour staging in colorectal cancer which showed no correlation with overall survival or disease recurrence. Our result thus cautions against the practice of equating an inverse relation of genetic markers with tumour staging to survival or disease recurrence.

The clinical use of all-trans-retinoic acid (ATRA) in the treatment of cancer is significantly hampered by the prompt emergence of resistance, believed to be caused by increased ATRA catabolism. Inhibitors of ATRA catabolism may therefore prove valuable for cancer therapy. Liarozole-fumarate is an anti-tumour drug that inhibits the cytochrome P450-dependent catabolism of ATRA. ATRA, but also its naturally occurring catabolites, 4-oxo-ATRA and 5,6-epoxy-ATRA, as well as its stereoisomers, 9-cis-RA and 13-cis-RA, show significant antiproliferative activity in MCF-7 human breast cancer cells. To further elucidate its mechanism of action, we investigated whether liarozole-fumarate was able to enhance the antiproliferative activity of ATRA catabolites and isomers. Liarozole-fumarate alone up to a concentration of 10(-6) M had no effect on MCF-7 cell proliferation. However, in combination with ATRA or the ATRA catabolites, liarozole-fumarate (10(-6) M) significantly enhanced their antiproliferative activity. On the contrary, liarozole-fumarate (10(-6) M) was not able to potentiate the antiproliferative activity of the ATRA stereoisomers, most probably because of the absence of cytochrome P450-dependent catabolism. Together, these findings show that liarozole-fumarate acts as a versatile inhibitor of retinoid catabolism in that it not only blocks the breakdown of ATRA, but also inhibits the catabolic pathway of 4-oxo-ATRA and 5,6-epoxy-ATRA, thereby enhancing their antiproliferative activity.

We assayed methyl-p-hydroxyphenyllactate esterase (MeHPLAase) activity in 63 cases of primary laryngeal squamous cell carcinoma. MeHPLAase activity did not show any correlation with oestrogen, progesterone and epidermal growth factor (EGF) receptor levels. No significant relationship was found between MeHPLAase activity and age, sex, tumour site, T classification, stage of disease and EGFR status, whereas a significant inverse relationship was found between enzymatic activity and neck lymph node positivity at presentation. The median value of MeHPLAase activity tended to be higher in tumours with low histopathological grade than in those with high histopathological grade. During the follow-up period (median 50 months, range 2-90 months) locoregional recurrences were observed in 31 out of 63 (49%) cases. At the end of the study, 27 out of 63 (43%) patients had died of cancer. Cox univariate analysis using MeHPLAase activity as a continuous covariate showed that the levels of enzymatic activity were inversely associated with the risk of death and relapse. Assuming the mean value of enzymatic activity as the cut-off value, we found a statistically significant relationship between high MeHPLAase activity and longer relapse-free and overall survival. MeHPLAase activity status retained its prognostic significance also in the lymph node-negative subgroup of patients. On multivariate analysis, both EGFR and MeHPLAase activity proved to be independent factors for predicting a short relapse and the overall survival.

We have noticed that extra domain A containing fibronectin (EDA(+)FN) is closely related to inflammatory diseases and accordingly investigated a selective adsorbent to remove EDA(+)FN, which may increase the severity of the symptoms, from the plasma of rheumatoid arthritis (RA) patients. Three types of heparin immobilized cellulose (HC) were prepared and their adsorptive properties were evaluated by batchwise adsorption tests. The results showed that EDA(+)FN was adsorbed more efficiently than total FN on HC. In particular HC in which heparin was immobilized to amino residue had an excellent capacity. The adsorption rates of EDA(+)FN, total FN and antithrombin-III were 89, 15 and 17% respectively. This adsorbent is extremely useful in selectively removing EDA(+)FN from plasma.

To investigate possible biochemical mechanisms underlying the "toxic gain of function" associated with polyglutamine expansions, the ability of guinea pig liver tissue transglutaminase to catalyze covalent attachments of various polyamines to polyglutamine peptides was examined. Of the polyamines tested, spermine is the most active substrate, followed by spermidine and putrescine. Formation of covalent cross links between polyglutamine peptides and polyamines yields high-M(r) aggregates--a process that is favored with longer polyglutamines. In the presence of tissue transglutaminase, purified glyceraldehyde-3-phosphate dehydrogenase (a key glycolytic enzyme that binds tightly to the polyglutamine domains of both huntingtin and dentatorubral-pallidoluysian atrophy proteins) is covalently attached to polyglutamine peptides in vitro, resulting in the formation of high-M(r) aggregates. In addition, endogenous glyceraldehyde-3-phosphate dehydrogenase of a Balb-c 3T3 fibroblast cell line overexpressing human tissue transglutaminase forms cross-links with a Q60 polypeptide added to the cell homogenate. Possibly, expansion of polyglutamine domains (thus far known to occur in the gene products associated with at least seven neurodegenerative diseases) leads to increased/aberrant tissue transglutaminase-catalyzed cross-linking reactions with both polyamines and susceptible proteins, such as glyceraldehyde-3-phosphate dehydrogenase. Formation of cross-linked heteropolymers may lead to deposition of high-M(r) protein aggregates, thereby contributing to cell death.

High-fat and low-fat diets were fed for 24 weeks to two strains of mice differing in susceptibility to the development of dystrophic cardiac calcinosis (DCC). The severity of DCC was assessed by histological examination and measurement of heart calcium concentrations. Plasma and renal tissue mineral levels were also measured. On the basis of macroscopic examination, feeding the high-fat diet did not influence the severity of DCC in the susceptible DBA/2 mice. Heart calcium concentrations were reduced in both strains; in DBA/2 mice more than in DCC-resistant C57BL/6 mice. Cardiac concentrations of magnesium and phosphorus were similar in the two strains of mice and unaffected by the amount of dietary fat. Kidney calcium concentrations were higher in the DBA/2 than in the C57BL/6 mice and the high-fat diet also reduced kidney calcium concentrations in the DBA/2 mice. The amount of dietary fat, at least during growth and maintenance, did not appear to affect the development of DCC in DBA/2 mice.

<h4>Background and objective</h4>Antithrombin III (ATIII) concentrates are employed as therapy for congenital or acquired deficiencies. These concentrates are obtained from Cohn's fraction IV1. To improve yields, purity and safety, our group developed a procedure to obtain a pasteurized ATIII concentrate from the supernatant of Cohn's fraction II + III including a highly efficient heparin affinity chromatography purification and pasteurization as a viral inactivation step.<h4>Design and methods</h4>Three steps of the manufacturing procedure (Cohn's fraction II + III precipitation, affinity chromatography and pasteurization) were selected to examine their efficacy in inactivating and/or removing the selected viruses.<h4>Results</h4>The industrial batches show a purity higher than 99% with approximately 95% native heparin binding ATIII. Only albumin and IgG could be detected at trace levels (0.07% and 0.16% of the total protein present, respectively). The specific activity of the product was approximately 6.65 IU/mg protein. Five viruses were spiked into the manufacturing starting materials and samples were collected at various points to determine the infection level of virus. The study showed a reduction factor (log 10) > or = 11.7 for HIV-1; > or = 8.1 for bovine herpes virus (analyzed as a model for herpes and hepatitis B viruses); > or = 8.1 for bovine diarrhea virus (model for hepatitis C and G) and > or = 6.0 for encephalomyocarditis virus (model for hepatitis A and other non-enveloped viruses).<h4>Interpretation and conclusions</h4>No biochemical alterations of the ATIII were detected in the final product. A high viral elimination capacity of the production process was demonstrated. So far, more than 32 million units of ATIII have been transfused in the form of this therapeutic concentrate without any detected seroconversion.

Antithrombin III (ATIII) and factor IX (FIX), two proteins from the clotting cascade, were investigated in parallel experiments, using capillary gel electrophoresis and capillary isoelectric focusing. The results from these experiments were compared with the results obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and slab gel isoelectric focusing. In the case of ATIII, capillary gel sieving showed comparable results to SDS-PAGE with the added advantage of the shorter time required for analysis. By optimizing capillary isoelectric focusing (cIEF), a separation of the ATIII isoforms was achieved. In the case of FIX, capillary gel electrophoresis (SDS-CE) of a FIX preparation gave similar results to those obtained by size-exclusion high-performance liquid chromatography and SDS-PAGE, but turned out to be less sensitive in detecting protein impurities at low concentrations. The microheterogeneity of this protein was shown by using cIEF.

HAP1 (huntingtin associated protein) has previously been found to interact with huntingtin (htt) in a glutamine length dependent manner and has been proposed to play a role in the cell specific neurodegeneration observed in Huntington's disease (HD). We have isolated mouse HAP1 (hap1) and have shown that expression is not enriched in areas specifically affected in HD. We have used the yeast two hybrid system to demonstrate that htt amino acids 171-230 are necessary for the hap1-htt binding and that hapl does not interact with the transgene exon 1 protein in a transgenic model of HD.

<h4>Background</h4>ATIII is decreased in sepsis and/or shock and its baseline value correlates with mortality. The efficacy of ATIII therapy on mortality was assessed in a selected group of patients admitted to the intensive care unit (ICU) in a double-blind, randomized, multicenter study.<h4>Methods</h4>120 patients admitted to the ICU with an ATIII concentration < 70% were randomized to receive ATIII (total dose 24000 units) or placebo treatment for 5 days; 56 patients had septic shock.<h4>Results</h4>ATIII concentrations in the treated group remained constant throughout the treatment period (range 97-102%). The Kaplan-Meier analysis showed no difference in overall survival between the two groups: 50 and 46% for ATIII and placebo, respectively. Septic shock and hemodynamic support were unbalanced in the two groups at admission. Therefore the Cox analysis was carried out after adjusting for these two variables. Treatment with ATIII decreases the risk of death with an odds ratio (OR) of 0.56. Of the covariates analyzed, septic shock and the baseline multiple organ failure score were negatively associated with survival and plasma activity level was positively associated with survival with an OR of 0.97 for each 1% increase in the ATIII plasma concentration at baseline.<h4>Conclusions</h4>The results of ATIII treatment in this population of patients suggests that replacement therapy reduces mortality in the subgroup of septic shock patients only.

<h4>Background</h4>HFE mutations are associated with hereditary haemochromatosis. However, a simple method capable of demonstrating the cis/trans arrangement of alleles is lacking, and linkage disequilibrium between HFE alleles and classic HLA loci is unknown. These are important issues as the pathogenic role of the mutations is not known.<h4>Aims</h4>To develop a simple method of genotyping HFE mutations suitable for clinical use in addition to large disease studies.<h4>Patients</h4>A total of 330 Caucasoid cadaveric organ donor controls were examined. Ten individuals previously HLA-H genotyped by polymerase chain reaction using restriction fragment length polymorphism (PCR-RFLP) were also examined to validate the method.<h4>Methods</h4>A simple polymerase chain reaction using sequence specific primers (PCR-SSP) capable of haplotyping the mutations was developed. HFE allele and haplotype frequencies and linkage disequilibrium with eight HLA class I and II loci were examined in the control population.<h4>Results</h4>27% and 19.7% of patients were positive for the 63D and 282Y alleles, respectively. No chromosome carried both 63D and 282Y. Linkage disequilibrium between 282Y and HLA-A*03 was confirmed, but was not straightforward: some A*03-associated alleles (DRB1*15, DQB1*06), but not all (B*07, Cw*0702), were associated with 282Y.<h4>Conclusions</h4>Linkage disequilibrium data suggest that an HLA-B*07 containing haplotype contains an element affording protection from haemochromatosis and may suggest the timing of the founder 282Y mutation.

Germline mutations in the RET proto-oncogene have been shown to be the underlying cause of multiple endocrine neoplasia type 2 (MEN 2A and 2B) and familial medullary thyroid carcinoma (FMTC). Some cases of sporadic medullary thyroid carcinoma (sporadic MTC) are reported to have specific codon 918, 883 and 768 mutations of the RET gene in tumor tissues. We examined RET gene mutations in 40 Japanese cases who had previously undergone surgery for sporadic MTC. DNA extracted from formalin-fixed tumor tissues and corresponding normal thyroid tissues or peripheral blood leukocytes was analyzed for mutations of exon 10, 11, 13, 14 and 16 of the RET gene by DNA sequencing and by mutation-specific restriction enzyme analysis. Germline RET point mutations were found in six of 40 cases (15%), cysteine residues at codon 618 in two, codon 634 in three and valine residue at codon 804 in one, and were newly identified as heritable MTC. Of the remaining 34 sporadic MTC cases, four (12%) had tumor-specific RET point mutations. Two were found in exon 16; one case showed an ATG to ACG (Met to Thr) mutation at codon 918, and the other showed two point mutations, ATG to ACG (Met to Thr) at codon 918 and GCA to GTA (Ala to Val) at codon 919 with loss of the wild-type allele, suggesting that both alleles at the RET locus were altered. The other two were found in exon 13; one case showed a CCG to TCG (Pro to Ser) mutation at codon 766 and the other showed a silent mutation, GTC to GTT (Val) at codon 778 with loss of the wild-type allele. There was no association of sporadic mutations with recurrence or prognosis in patients with sporadic MTC. The low rate of somatic RET mutation at codon 918 in our sporadic MTC suggests that as yet unknown factors may be involved. Genetic alterations in both alleles may have an important role in small fraction of sporadic MTCs.

Each porphyria results from decreased activity of one of the enzymes of haem biosynthesis. The molecular basis of enzyme deficiencies in acute intermittent porphyria (AIP), variegate porphyria (VP) and congenital erythropoietic porphyria (CEP) is outlined. All three conditions show extensive allelic heterogeneity. In the autosomal dominant disorders, AIP and VP, no genotype/phenotype correlations have been demonstrated, and the explanation for their low clinical penetrance remains uncertain. In AIP and VP, mutational analysis is superior to biochemical methods for screening families for latent porphyria. In the autosomal recessive condition, CEP, there is some genotype/phenotype correlation--one common mutation (C73R) being associated with severe disease in homozygotes. Porphyria cutanea tarda (PCT) is not a simple monogenic disorder. Patients appear to have an inherited susceptibility to inactivation of hepatic uroporphyrinogen decarboxylase (UROD) as part of a response to hepatocyte injury by alcohol, HCV and other agents. Inherited factors that, in combination, may predispose to PCT include mutations in the UROD gene, present in about 20% of patients, and the C282Y mutation in the haemochromatosis (HFE) gene.

Huntington's disease (HD) is caused by CAG triplet repeat expansion in IT15 which leads to polyglutamine stretches in the HD protein product, huntingtin. The pathological hallmark of HD is the degeneration of subsets of neurons, primarily those in the striatum and neocortex. Specific morphological markers of affected cells have not been identified in patients with HD, although a unique itranuclear inclusion was recently reported in neurons of transgenic animals expressing a construct encoding the N-terminal part (including the glutamine repeat) of huntingtin (Davies et al., 1997). In order to understand the importance of this finding, we sought for comparable nuclear abnormalities in autopsy material from patients with HD. In all 20 HD cases examined, anti-ubiquitin and N-terminal huntingtin antibodies identified itranuclear inclusions in neurons and the frequency of these lesions correlated with the length of the CAG repeat in IT15. In addition, examination of material from the related HD-like triplet repeat disorder, dentatorubral and pallidoluysian atrophy, also revealed intranuclear neuronal inclusions. These findings suggest that intranuclear inclusions containing protein aggregates may be common feature of the pathogenesis of glutamine repeat neurodegenerative disorders.

Genetic haemochromatosis (GH) is the most common, autosomal recessive disorder in Northern Europe. The studies which led to the identification of the HFE gene are described. In the UK over 90% of patients with GH are homozygous for the C282Y mutation of this gene. This mutation is confined to populations of European origin. The significance of another mutation, H63D, in causing iron overload is less certain. Preliminary studies on the localization of the protein and the effects of the mutations are described. Genetic testing and the measurement of iron status now provide the means to allow for widespread testing for the prevention of iron overload and its consequences. However, questions remain about the clinical penetrance of GH.

<h4>Objectives</h4>To investigate articular cartilage collagen network, thickness of birefringent cartilage zones, and glycosaminoglycan concentration in macroscopically normal looking knee joint cartilage of young beagles subjected to experimental slowly progressive osteoarthritis (OA).<h4>Methods</h4>OA was induced by a tibial 30 degree valgus osteotomy in 15 female beagles at the age of 3 months. Fifteen sisters were controls. Cartilage specimens were collected seven (Group 1) and 18 months (Group 2) postoperatively. Collagen induced optical path difference and cartilage zone thickness measurements were determined from histological sections of articular cartilage with smooth and intact surface by computer assisted quantitative polarised light microscopy. Volume density of cartilage collagen fibrils was determined by image analysis from transmission electron micrographs and content of glycosaminoglycans by quantitative digital densitometry from histological sections.<h4>Results</h4>In the superficial zone of the lateral tibial and femoral cartilage, the collagen induced optical path difference (birefringence) decreased by 19 to 71% (p < 0.05) seven months postoperatively. This suggests that severe superficial collagen fibril network deterioration took place, as 18 months postoperatively, macroscopic and microscopic OA was present in many cartilage areas. Thickness of the uncalcified cartilage increased while the superficial zone became thinner in the same sites. In operated dogs, glycosaminoglycan content first increased (Group 1) in the lateral tibial condyle and then decreased (Group 2) (p < 0.05).<h4>Conclusion</h4>In this OA model, derangement of the superficial zone collagen network was the probable reason for birefringence reduction. This change occurred well before macroscopic OA.