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Screening for genetic variants that predispose individuals or their offspring to disease may be performed at the general population level or may instead be targeted at the relatives of previously identified carriers. The latter strategy has come to be known as "cascade genetic screening." Since the carrier risk of close relatives of known carriers is generally higher than the population risk, cascade screening is more efficient than population screening, in the sense that fewer individuals have to be genotyped per detected carrier. The efficacy of cascade screening, as measured by the overall proportion of carriers detected in a given population, is, however, lower than that of population-wide screening, and the respective inclusion rates vary according to the population frequency and mode of inheritance of the predisposing variants. For dominant mutations, we have developed equations that allow the inclusion rates of cascade screening to be calculated in an iterative fashion, depending upon screening depth and penetrance. For recessive mutations, we derived only equations for the screening of siblings and the children of patients. Owing to their mathematical complexity, it was necessary to study more extended screening strategies by simulation. Cascade screening turned out to result in low inclusion rates (<1%) when aimed at the identification of heterozygous carriers of rare recessive variants. Considerably higher rates are achievable, however, when screening is performed to detect covert homozygotes for frequent recessive mutations with reduced penetrance. This situation is exemplified by hereditary hemochromatosis, for which up to 40% of at-risk individuals may be identifiable through screening of first- to third-degree relatives of overt carriers (i.e., patients); the efficiency of this screening strategy was found to be approximately 50 times higher than that of population-wide screening. For dominant mutations, inclusion rates of cascade screening were estimated to be higher than for recessive variants. Thus, some 80% of all carriers of the factor V Leiden mutation would be detected if screening were to be targeted specifically at first- to third-degree relatives of patients with venous thrombosis. The relative cost efficiency of cascade as compared with population-wide screening (i.e., the overall savings in the extra managerial cost of the condition) is also likely to be higher for dominant than for recessive mutations. This notwithstanding, once screening has become cost-effective at the population level, it can be expected that cascade screening would only transiently represent an economically viable option.

Zn-alpha(2)-glycoprotein (ZAG) is a member of the major histocompatibility complex (MHC) class I family of proteins and is identical in amino acid sequence to a tumor-derived lipid-mobilizing factor associated with cachexia in cancer patients. ZAG is present in plasma and other body fluids, and its natural function, like leptin's, probably lies in lipid store homeostasis. X-ray crystallography has revealed an open groove between the helices of ZAG's alpha(1) and alpha(2) domains, containing an unidentified small ligand in a position similar to that of peptides in MHC proteins (Sanchez, L. M., Chirino, A. J., and Bjorkman, P. J. (1999) Science 283, 1914-1919). Here we show, using serum-derived and bacterial recombinant protein, that ZAG binds the fluorophore-tagged fatty acid 11-(dansylamino)undecanoic acid (DAUDA) and, by competition, natural fatty acids such as arachidonic, linolenic, eicosapentaenoic, and docosahexaenoic acids. Other MHC class I-related proteins (FcRn, HFE, HLA-Cw*0702) showed no such evidence of binding. Fluorescence and isothermal calorimetry analysis showed that ZAG binds DAUDA with K(d) in the micromolar range, and differential scanning calorimetry showed that ligand binding increases the thermal stability of the protein. Addition of fatty acids to ZAG alters its intrinsic (tryptophan) fluorescence emission spectrum, providing a strong indication that ligand binds in the expected position close to a cluster of exposed tryptophan side chains in the groove. This study therefore shows that ZAG binds small hydrophobic ligands, that the natural ligand may be a polyunsaturated fatty acid, and provides a fluorescence-based method for investigating ZAG-ligand interactions.

Huntingtin is a 350-kilodalton protein of unknown function that is mutated in Huntington's disease (HD), a neurodegenerative disorder. The mutant protein is presumed to acquire a toxic gain of function that is detrimental to striatal neurons in the brain. However, loss of a beneficial activity of wild-type huntingtin may also cause the death of striatal neurons. Here we demonstrate that wild-type huntingtin up-regulates transcription of brain-derived neurotrophic factor (BDNF), a pro-survival factor produced by cortical neurons that is necessary for survival of striatal neurons in the brain. We show that this beneficial activity of huntingtin is lost when the protein becomes mutated, resulting in decreased production of cortical BDNF. This leads to insufficient neurotrophic support for striatal neurons, which then die. Restoring wild-type huntingtin activity and increasing BDNF production may be therapeutic approaches for treating HD.

<h4>Background</h4>Receptor protein tyrosine phosphatase rho (RPTPrho, gene symbol PTPRT) is a member of the type IIB RPTP family. These transmembrane molecules have been linked to signal transduction, cell adhesion and neurite extension. The extracellular segment contains MAM, Ig-like and fibronectin type III domains, and the intracellular segment contains two phosphatase domains. The human RPTPrho gene is located on chromosome 20q12-13.1, and the mouse gene is located on a syntenic region of chromosome 2. RPTPrho expression is restricted to the central nervous system.<h4>Results</h4>The cloning of the mouse cDNA, identification of alternatively spliced exons, detection of an 8 kb 3'-UTR, and the genomic organization of human and mouse RPTPrho genes are described. The two genes are comprised of at least 33 exons. Both RPTPrho genes span over 1 Mbp and are the largest RPTP genes characterized. Exons encoding the extracellular segment through the intracellular juxtamembrane 'wedge' region are widely spaced, with introns ranging from 9.7 to 303.7 kb. In contrast, exons encoding the two phosphatase domains are more tightly clustered, with 15 exons spanning approximately 60 kb, and introns ranging in size from 0.6 kb to 13.1 kb. Phase 0 introns predominate in the intracellular, and phase 1 in the extracellular segment.<h4>Conclusions</h4>We report the first genomic characterization of a RPTP type IIB gene. Alternatively spliced variants may result in different RPTPrho isoforms. Our findings suggest that RPTPrho extracellular and intracellular segments originated as separate modular proteins that fused into a single transmembrane molecule during a later evolutionary period.

Classical midgut carcinoids are rare intestinal neuroendocrine tumors that often present with metastases at diagnosis. In contrast to foregut carcinoids, midgut carcinoids are not related to the multiple endocrine neoplasia type 1 syndrome, and the mechanisms involved in their tumorigenesis are unknown. Eight classical midgut carcinoids were analyzed by genome-wide screening for loss of heterozygosity. Deletions on chromosome 18 were found in 88% of the tumors. DNA sequencing and immunohistochemical staining for Smad4/DPC4, which often is homozygously mutated in pancreatic and colon carcinomas, revealed no aberrations. In 1 tumor, a region telomeric to the Smad4/DPC4/DCC genes at 18q21 was deleted. Other chromosomes were affected in 3 lesions only. The high frequency of chromosome 18 deletions strongly indicates a genetic alteration of importance in classical midgut carcinoid tumorigenesis, apparently not involving the Smad4/DPC4 gene.

Netrins are a family of secreted proteins that function as chemotropic axon guidance cues during neural development. Here we demonstrate that netrin-1 continues to be expressed in the adult rat spinal cord at a level similar to that in the embryonic CNS. In contrast, netrin-3, which is also expressed in the embryonic spinal cord, was not detected in the adult. In situ hybridization analysis demonstrated that cells in the white matter and the gray matter of the adult spinal cord express netrin-1. Colocalization studies using the neuronal marker NeuN revealed that netrin-1 is expressed by multiple classes of spinal interneurons and motoneurons. Markers identifying glial cell types indicated that netrin-1 is expressed by most, if not all, oligodendrocytes but not by astrocytes. During neural development, netrin-1 has been proposed to function as a diffusible long-range cue for growing axons. We show that in the adult spinal cord the majority of netrin-1 protein is not freely soluble but is associated with membranes or the extracellular matrix. Fractionation of adult spinal cord white matter demonstrated that netrin-1 was absent from fractions enriched for compact myelin but was enriched in fractions containing periaxonal myelin and axolemma, indicating that netrin-1 protein may be localized to the periaxonal space. These findings suggest that in addition to its role as a long-range guidance cue for developing axons, netrin may have a short-range function associated with the cell surface that contributes to the maintenance of appropriate neuronal and axon-oligodendroglial interactions in the mature nervous system.

Mitochondrial defects, which occur in the brain of late-stage Huntington's disease (HD) patients, have been proposed to underlie the selective neuronal loss in the disease. To shed light on the possible role of mitochondrial energy impairment in the early phases of HD pathophysiology, we carried out Golgi impregnation and quantitative histochemical/biochemical studies in HD full-length cDNA transgenic mice that were symptomatic but had not developed to a stage in which neuronal loss could be documented. Golgi staining showed morphologic abnormalities that included a significant decrease in the number of dendritic spines and a thickening of proximal dendrites in striatal and cortical neurons. In contrast, measurements of mitochondrial electron transport Complexes I-IV did not reveal changes in the striatum and cerebral cortex in these mice. Examination of the neostriatum and cerebral cortex in human presymptomatic and pathological Grade 1 HD cases also showed no change in the activity of mitochondrial Complexes I-IV. These data suggest that dendritic alterations precede irreversible cell loss in HD, and that mitochondrial energy impairment is a consequence, rather than a cause, of early neuropathological changes.

<h4>Background</h4>First considered as a polymorphism of the HFE gene, the H63D mutation is now widely recognised as a haemochromatosis associated allele. But few H63D homozygotes with clinical manifestations of hereditary haemochromatosis (HH) have been reported. Concurrently, an increasing number of genes have been shown to interact with HFE in iron metabolism.<h4>Aims</h4>To describe the clinical expression of iron overload (IO) associated with H63D homozygosity, and search for potential genetic modifiers (within the HFE or other genes) that could explain the variability of the phenotypes.<h4>Patients and methods</h4>We retrospectively analysed the clinical phenotype of 56 H63D homozygotes referred for a personal or family history of IO. For each subject we examined intragenic HFE haplotypes and transferrin receptor (TfR) gene polymorphisms and searched for the Y250X mutation on the TFR2 gene. Additionally, we sequenced the HFE gene of H63D homozygotes with HH.<h4>Results</h4>Fifty of 56 subjects had biological and/or clinical abnormalities of iron metabolism. Up to two thirds of patients (n=34) had no acquired cause of IO. Among these, 12 had a phenotypic diagnosis of HH. In the iron loaded group there was a strong prevalence of male patients. No correlation was found between the potential genetic modifiers and phenotypes. No additional mutation of HFE was identified.<h4>Conclusion</h4>The variable phenotypes associated with H63D homozygosity do not appear to be linked to other HFE mutations, to the TFR2 Y250X mutation, or to HFE or TfR gene intragenic polymorphisms. The exact role of H63D homozygosity in IO and HH needs to be further investigated in unselected populations.

Transfer of CD45RB(high) CD4+ T cells to immune-deficient mice in the absence of regulatory T cells leads to a Th1-mediated colitis. In this study, we show that intestinal inflammation is characterized by a 15-fold increase in the number of CD134L+ (OX40L+)-activated DC in the mesenteric lymph nodes (MLNs) compared with BALB/c mice. This was important functionally, as administration of an anti-CD134L mAb inhibited the proliferation of T cells in the MLNs as well as their expression of the gut-homing integrin alpha(4)beta(7). Most importantly, the anti-CD134L mAb completely blocked development of colitis. Surprisingly, CD134L was found to be expressed by a proportion of dendritic cells (DC) in the MLNs of unreconstituted SCID mice, suggesting that CD134L can be induced on DC in the absence of T cell-derived signals. These results indicate that some DC in the MLNs of SCID mice express an activated phenotype and that CD134L expression by these cells is involved in the development of colitis induced by T cell transfer. Accumulation of CD134L+ DC was inhibited by cotransfer of regulatory T cells, suggesting that inhibition of the accumulation of activated DC is one mechanism by which these cells prevent immune pathology.

Translation elongation factor 1delta (EF-1delta) is hyperphosphorylated in various mammalian cells infected with alpha-, beta- and gammaherpesviruses and EF-1delta modification is mediated by viral protein kinases, including UL13 of herpes simplex virus type 1 and UL97 of human cytomegalovirus. In this study, the following is reported. (i) BGLF4 encoded by the prototype gammaherpesvirus Epstein-Barr virus was purified as a fusion protein that was labelled with [gamma-(32)P]ATP and labelling was eliminated by phosphatase. (ii) The ratio of the hyperphosphorylated form of human EF-1delta was increased both in Sf9 cells after infection with baculoviruses expressing GST-BGLF4 fusion proteins and in COS-7 cells after transfection with a BGLF4 expression plasmid. These results indicate that purified BGLF4 possesses protein kinase activity and mediates EF-1delta hyperphosphorylation. These data also support the hypothesis that the protein kinases that are conserved by herpesviruses universally mediate EF-1delta modification in mammalian cells.

The nucleotide sequences of the 27,939-bp-long upstream and 9,448-bp-long downstream regions of the carAaAaBaBbCAc(ORF7)Ad genes of carbazole-degrading Pseudomonas sp. strain CA10 were determined. Thirty-two open reading frames (ORFs) were identified, and the car gene cluster was consequently revealed to consist of 10 genes (carAaAaBaBbCAcAdDFE) encoding the enzymes for the three-step conversion of carbazole to anthranilate and the degradation of 2-hydroxypenta-2,4-dienoate. The high identities (68 to 83%) with the enzymes involved in 3-(3-hydroxyphenyl)propionic acid degradation were observed only for CarFE. This observation, together with the fact that two ORFs are inserted between carD and carFE, makes it quite likely that the carFE genes were recruited from another locus. In the 21-kb region upstream from carAa, aromatic-ring-hydroxylating dioxygenase genes (ORF26, ORF27, and ORF28) were found. Inductive expression in carbazole-grown cells and the results of homology searching indicate that these genes encode the anthranilate 1,2-dioxygenase involved in carbazole degradation. Therefore, these ORFs were designated antABC. Four homologous insertion sequences, IS5car1 to IS5car4, were identified in the neighboring regions of car and ant genes. IS5car2 and IS5car3 constituted the putative composite transposon containing antABC. One-ended transposition of IS5car2 together with the 5' portion of antA into the region immediately upstream of carAa had resulted in the formation of IS5car1 and ORF9. In addition to the insertion sequence-dependent recombination, gene duplications and presumed gene fusion were observed. In conclusion, through the above gene rearrangement, the novel genetic structure of the car gene cluster has been constructed. In addition, it was also revealed that the car and ant gene clusters are located on the megaplasmid pCAR1.

SOX9 is a transcription factor that activates type II procollagen (Col2a1) gene expression during chondrocyte differentiation. Glucocorticoids are also known to promote chondrocyte differentiation via unknown molecular mechanisms. We therefore investigated the effects of a synthetic glucocorticoid, dexamethasone (DEX), on Sox9 gene expression in chondrocytes prepared from rib cartilage of newborn mice. Sox9 mRNA was expressed at high levels in these chondrocytes. Treatment with DEX enhanced Sox9 mRNA expression within 24 h and this effect was observed at least up to 48 h. The effect of DEX was dose dependent, starting at 0.1 nM and maximal at 10 nM. The half life of Sox9 mRNA was approximately 45 min in the presence or absence of DEX. Western blot analysis revealed that DEX also enhanced the levels of SOX9 protein expression. Treatment with DEX enhanced Col2a1 mRNA expression in these chondrocytes and furthermore, DEX enhanced the activity of Col2-CAT (chloramphenicol acetyltransferase) construct containing a 1.6 kb intron fragment where chondrocyte-specific Sry/Sox- consensus sequence is located. The enhancing effect of DEX was specific to SOX9, as DEX did not alter the levels of Sox6 mRNA expression. These data suggest that DEX promotes chondrocyte differentiation through enhancement of SOX9.

Colorectal cancers with DNA mismatch repair (MMR) gene mutations characteristically display a high rate of replication errors in simple repetitive sequences detectable as microsatellite instability (MSI). Most are the result of somatic MMR dysfunction; however, a subset are caused by germline mutations. The availability of commercial antibodies for MSH2 and MLH1 [corrected] offers an alternative strategy to molecular methods for identifying MMR deficient cancers. To evaluate immunohistochemistry, MLH1 and MSH2 expression was studied using monoclonal antibodies in formalin fixed, paraffin wax embedded cancers. The immunohistochemical staining patterns of 23 cancers displaying MSI, including four cases with germline mutations, were compared with 23 microsatellite stable (MSS) cancers. All MSS cancers exhibited staining with both antibodies. Twenty two of the MSI cases showed absent MMR expression with either anti-MSH2 or anti-MLH1 [corrected]. The high sensitivity and predictive value of immunohistochemistry in detecting MMR deficiency offers a method of discriminating between MSI and MSS cancers caused by MSH2 and MLH1 [corrected] dysfunction. The application and suitability of immunohistochemistry for the detection of MSI and as a strategy for prioritising the mutational analysis of MMR genes in routine clinical practice is discussed.

p53 protein expression and oestrogen and progesterone receptor status in invasive ductal breast carcinomas The p53 protein expression and oestrogen and progesterone receptors status was investigated in correlation to the grade of malignancy of primary breast carcinomas. Our material constituted imprints from surgical biopsies of 75 invasive ductal breast cancer cases. The p53 protein expression was investigated immunocytologically using the monoclonal antibody p53 DO-7 (DAKO). A biochemical DCC method was applied for the detection of oestrogen and progesterone receptors for all tumours. Fifty-one percent of breast cancer cases were p53 protein positive. A statistically significant association of p53 protein expression and high tumour grade was found (chi2=23.72, d.f.=2, P < 0.001). A statistically significant association was also found between oestrogen and progesterone receptor positive cases and the grade of malignancy (P < 0.001). A negative association between p53 protein expression and oestrogen (ER) and progesterone receptors (PgR) positivity was found. From our results it appears that it is possible to distinguish from grade II tumours two subgroups of cases, one with low malignancy potential and p53 (-), ER (+), PgR (+), and another subgroup with high malignancy potential and phenotype p53 (+), ER (-), PgR (-). The last subset of patients could actually benefit from adjuvant therapy.

<h4>Purpose</h4>This study was designed to investigate the suppressive effects of antithrombin (AT)III on inflammatory reactions during endotoxin-induced uveitis (EIU) in rats by studying leukocyte-endothelium interactions.<h4>Methods</h4>EIU was induced in Lewis rats by footpad injection of lipopolysaccharide (LPS). ATIII was administered immediately after or at 6 hours after LPS injection. Its suppressive effects on inflammatory leukocyte behavior were evaluated in vivo with acridine orange digital fluorography. Clinical signs of inflammation were also examined, and aqueous humor (AH) was collected to evaluate leukocyte infiltration and protein leakage. In a separate experiment, P-selectin mRNA expression was studied in the iris-ciliary body (ICB) and the retina.<h4>Results</h4>After treatment with ATIII, leukocyte rolling was substantially inhibited along the retinal veins, suppressing subsequent leukocyte infiltration into the vitreous cavity. Similarly, leukocyte infiltration and protein leakage into the AH were significantly reduced with ATIII treatment. The clinical grade of EIU was substantially lower in ATIII-treated rats. In addition, delayed administration of ATIII after EIU induction significantly attenuated these inflammatory reactions. The levels of P-selectin mRNA expression in both ICB and retina, which were upregulated after LPS injection, were substantially lower in the ATIII-treated rats.<h4>Conclusions</h4>ATIII treatment significantly inhibited inflammatory reactions induced with LPS. Its suppressive effects on P-selectin expression could contribute to the attenuation of leukocyte infiltration, possibly by inhibiting leukocyte rolling. The current findings suggest that ATIII may have a role in the management of patients with uveitis.

"It has been commented by someone that 'polyoma' is an adjective composed of a prefix and suffix, with no root between--a meatless linguistic sandwich" (C. J. Dawe). The very name "polyomavirus" is a vague mantel: a name given before our understanding of these viral agents was clear but implying a clear tumor life-style, as noted by the late C. J. Dawe. However, polyomavirus are not by nature tumor-inducing agents. Since it is the purpose of this review to consider the natural function of middle T antigen (MT), encoded by one of the seemingly crucial transforming genes of polyomavirus, we will reconsider and redefine the virus and its MT gene in the context of its natural biology and function. This review was motivated by our recent in vivo analysis of MT function. Using intranasal inoculation of adult SCID mice, we have shown that polyomavirus can replicate with an MT lacking all functions associated with transformation to similar levels to wild-type virus. These observations, along with an almost indistinguishable replication of all MT mutants with respect to wild-type viruses in adult competent mice, illustrate that MT can have a play subtle role in acute replication and persistence. The most notable effect of MT mutants was in infections of newborns, indicating that polyomavirus may be highly adapted to replication in newborn lungs. It is from this context that our current understanding of this well-studied virus and gene is presented.

<h4>Background</h4>The occurrence of mania during antidepressant treatment is a key issue in the clinical management of bipolar disorder (BP). The serotonin transporter (5-HTT) is the selective site of action of most proserotonergic compounds used to treat bipolar depression. The 5-HTT gene (SLC6A4) has 2 known polymorphisms. The aim of this study was to investigate the role of the SLC6A4 variants in the pathogenesis of antidepressant-induced mania in BP.<h4>Methods</h4>Twenty-seven patients with a DSM-IV diagnosis of BP I or II, with at least 1 manic or hypomanic episode induced by treatment with proserotonergic antidepressants (IM+ group), were compared with 29 unrelated, matched patients with a diagnosis of BP I or II, who had been exposed to proserotonergic antidepressants without development of manic or hypomanic symptoms (IM- group). The 2 known polymorphisms of the SLC6A4 were genotyped, and allelic and genotypic association analyses were performed.<h4>Results</h4>With respect to the polymorphism in the promoter region (5HTTLPR), IM+ patients had an excess of the short allele (n = 34 [63%]) compared with IM- patients (n = 17 [29%]) (chi(2)(1), 12.77; P <.001). The genotypic association analysis showed a higher rate of homozygosity for the short variant in the IM+ group (n = 10 [37%]) than in the IM- group (n = 2 [7%]) and a lower rate of homozygosity for the long variant in the IM+ group (n = 3 [11%]) compared with the IM- group (n = 14 [48%]) (chi(2)(2), 12.43; P =.002). No associations were found for the polymorphism involving a variable number of tandem repeats.<h4>Conclusion</h4>If these results are replicated, the 5HTTLPR polymorphism may become an important predictor of abnormal response to medication in patients with BP.

The membrane receptors DCC and UNC5H have been shown to be crucial for axon guidance and neuronal migration by acting as receptors for netrin-1. DCC has also been proposed as a dependence receptor inducing apoptosis in cells that are beyond netrin-1 availability. Here we show that the netrin-1 receptors UNC5H (UNC5H1, UNC5H2, UNC5H3) also act as dependence receptors. UNC5H receptors induce apoptosis, but this effect is blocked in the presence of netrin-1. Moreover, we demonstrate that UNC5H receptors are cleaved in vitro by caspase in their intracellular domains. This cleavage may lead to the exposure of a fragment encompassing a death domain required for cell death induction in vivo. Finally, we present evidence that during development of the nervous system, the presence of netrin-1 is crucial to maintain survival of UNC5H- and DCC-expressing neurons, especially in the ventricular zone of the brainstem. Altogether, these results argue for a role of netrin-1 during the development of the nervous system, not only as a guidance cue but as a survival factor via its receptors DCC and UNC5H.

Severe iron overload usually develops in patients with hereditary hemochromatosis (HHC), but variability in the phenotypic expression of the disease has been reported. This study assessed whether tumor necrosis factor alpha (TNF-alpha) plays a role in phenotypic expression of HHC. Sixty-four patients with HHC and 172 healthy volunteers (controls) were studied. Release of TNF-alpha from stimulated peripheral blood monocytes was measured by enzyme-linked immunosorbent assay, and 308 and 238 TNF-alpha polymorphisms were detected with polymerase chain reaction and restriction fragment-length polymorphism analysis. The relation between TNF-alpha polymorphisms and clinical expression of HHC was evaluated. Patients with HHC released less TNF-alpha than controls, but the difference was significant only in homozygotes for the C282Y mutation. The prevalence of the 308 TNF-alpha polymorphism was similar in patients and controls, whereas the prevalence of the 238 polymorphic allele was significantly lower in patients (3% versus 16%; P =.002). A lower prevalence of cirrhosis was observed in patients with TNF-alpha polymorphism than in those without it (4 of 15 [27%] versus 28 of 49 [57%]), but the difference was not significant (P =.07). In nonhomozygotes for the C282Y mutation, severe liver siderosis was less prevalent in patients with the 308 polymorphism than in those without it (P =.05). Alanine aminotransferase (ALT) values were significantly lower in patients with TNF-alpha polymorphism (P =.006), even when patients with other hepatotoxic factors were excluded. Multivariate analysis showed that TNF-alpha polymorphism was independently associated with ALT values (P =.0008 and P =.045, respectively, in homozygotes and nonhomozygotes for the C282Y mutation) and siderosis in nonhomozygotes (P =.047). Thus, TNF-alpha appears to play a role in HHC by modulating the severity of liver damage. (Blood. 2001;97:3707-3712)

In Huntington's Disease (HD), the huntingtin protein (Htt) includes an expanded polyglutamine domain. Since mutant Htt concentrates in the nucleus of affected neurons, we have inquired whether normal Htt (Q16--23) is also able to access the nucleus. We observe that a major pool of normal full-length Htt of HeLa cells is anchored to endosomes and also detect RNase-sensitive nuclear foci which include a 70-kDa N-terminal Htt fragment. Agents which damage DNA trigger caspase-3-dependent cleavage of Htt and dramatically relocate the 70 kDa fragment to the nucleoplasm. Considering that polyglutamine tracts stimulate caspase activation, mutant Htt is therefore poised to enter the nucleus. These considerations help rationalize the nuclear accumulation of Htt which is characteristic of HD and provide a first example of involvement of caspase cleavage in release of membrane-bound proteins which subsequently enter the nucleus.

Analyses of cancer incidence data in the United States and Western Europe revealed steadily rising rates over the past decades of adenocarcinomas of the esophagus and gastric cardia. Genetic information on gastric cardia adenocarcinoma and its preneoplasias is sparse. We have used comparative genomic hybridization to obtain a genome-wide overview of 20 archival gastric cardia adenocarcinomas and 10 adjacent preneoplastic lesions (4 metaplasias, 1 low-grade dysplasia, 5 high-grade dysplasias). Multiple genetic alterations were discriminated in all adenocarcinomas. Frequent loss (> or =25% of all tumors) was detected, in decreasing order of frequency, on 5q, 18q, 4q, 3p, 9p, 2q, 11q, 14q, 21q, 4p, 9q, 16q, 1p, and 8p. Frequent gain (> or =25% of all tumors) was disclosed, in decreasing order of frequency, on 20q, 7p, 8q, 1q, 7q, 20p, 17q, 13q, Xp, 6q, 8p, 19q, 5p, 6p, and Xq. Loss of the Y chromosome was found in 60% of male cases. High level amplification was frequently (>10% of all tumors) detected on 7q21, 8p22, 12p11.2, 17q12-q21, and 19q13.1-q13.2. The precursor lesions showed multiple aberrations in all high-grade dysplasias, whereas few genetic changes were discerned in LGD and metaplasias. High level amplifications were also found in high-grade dysplasias, ie, on 7q21, 8p22, and 17q12-q21. Moreover, the percentage of aberrations was not significantly different for invasive carcinomas or high-grade dysplasias. Approximately 70% of the precursor aberrations were also present in the adjacent carcinoma. Minimal overlapping regions in the preneoplasias included loss on 18q12-q21 and gains on 8q23 and 17q12-q21, suggesting involvement of genes residing in these regions. In conclusion, we have (i) created a map of genetic alterations in gastric cardia adenocarcinomas and (ii) provided evidence for the presence of a metaplasia-dysplasia-carcinoma sequence in this poorly understood type of cancer.

<h4>Background</h4>A multitude of recommendations exist for laboratory assays to monitor the pace and endpoints of phlebotomy therapy for hemochromatosis. All of these recommendations rely on an assessment of storage iron to guide treatment, and none have been prospectively evaluated.<h4>Study design and methods</h4>Nine consecutive patients underwent serial monitoring of Hb, MCV, transferrin saturation, and ferritin during weekly phlebotomy to deplete iron stores (induction therapy) and less frequent sessions to prevent iron reaccumulation (maintenance therapy). Changes in MCV and Hb were used to guide the pace of phlebotomy over a median of 7 years of follow-up.<h4>Results</h4>During induction therapy, the MCV increased transiently because of reticulocytosis and then stabilized for a prolonged period before decreasing more sharply, which reflected iron-limited erythropoiesis. Iron depletion was achieved after a median of 38 phlebotomies and removal of 9.0 g of iron. Maintenance phlebotomy was targeted to maintain the MCV at 5 to 10 percent below prephlebotomy values and the Hb at >13 g per dL. Transferrin saturation fluctuated considerably during treatment, but remained below 35 percent during MCV-guided maintenance therapy. Ferritin values were not useful guides to the pace of phlebotomy. The median maintenance therapy phlebotomy interval was 7.5 weeks (range, 6-16), which corresponded to an average daily iron removal of 35 to 67 microg per kg. Most patients showed evidence of iron reaccumulation at phlebotomy intervals of 8 weeks or more.<h4>Conclusion</h4>The MCV is an inexpensive, precise, physiologic indicator of erythropoietic iron availability. When used in conjunction with the Hb, it is a clinically useful guide to the pace of phlebotomy therapy for hemochromatosis.

<h4>Background</h4>Hereditary hemochromatosis (HH) is a HFE gene-linked disorder affecting 1 of 200 to 400 persons in white populations. It has been proposed that patients with a hematologic malignancy who are receiving frequent RBC transfusions should be screened for HFE mutations. This would identify C282Y homozygotes, who have a high risk of developing severe iron overload.<h4>Study design and methods</h4>DNA samples from 128 controls and 23 adult long-term survivors of acute myeloid leukemia (AML) treated at the Oulu University Hospital (Oulu, Finland) from 1987 to 2000 were examined for the presence of the C282Y and H63D mutations in HFE. All the patients were severely iron-overloaded, as determined from high serum ferritin values and/or increased storage iron in bone marrow. Phlebotomies were performed in five patients because of the symptoms of iron overload. DNA extracted from the blood was used to amplify HFE gene fragments by the PCR method, after which the amplification products were digested with restriction endonucleases SnaB I and Bcl I, and the restriction fragments were analyzed on agarose gels.<h4>Results</h4>No chromosomes with the C282Y mutation were found among the AML patients, and 5 patients (21.7%) were heterozygous for the H63D mutation. In the control group, 13 persons (10.2%) were heterozygous for the C282Y mutation and 26 (20.3%) for the H63D mutation, including 3 C282Y/H63D double heterozygotes.<h4>Conclusion</h4>HFE mutations do not account for the harmful iron overload that develops in AML patients who receive large quantities of RBC concentrates after intensive chemotherapy.

<h4>Objective</h4>To evaluate the role of telomere and DCC in tumor transformation and progression.<h4>Methods</h4>Telomere length and DCC gene mRNA expression were examined by southern blot hybridization and RT-PCR analysis in 46 adenomas of large intestine, 62 cancers of large intestine and corresponding normal mucosa.<h4>Results</h4>Shortening of the telomere was present in the tissues of 41.3% of the adenomas and 53.2% of the cancers, and their average TRF lengths were significantly shorter than those of corresponding normal mucosa(P<0.05, P<0.01), whereas the telomere elongation was only detected in 4.4% and 6.5% of the adenomas and cancers respectively. In addition, the average telomere length in colon carcinomas was also shorter than that in rectal carcinomas. Moreover, the average telomere lengths of the colorectal cancer mucosa became shorter with age. The rates of DCC mRNA expression deletion were 34.8% and 62.9% in the tissues of adenomas and cancers respectively. The DCC mRNA expression deletion occurred more frequently in poorly differentiated and Dukes C, D carcinomas than in well-differentiated and Dukes A, B carcinomas (P<0.05, P<0.01). However, no significant correlation was found between the length of telomere and the deletion of DCC mRNA expression in the cancers of large intestine.<h4>Conclusion</h4>The telomere shortening and DCC mRNA deletion may represent the biologic behavior of transformation and development of the large intestine cancers.

The mammalian striatum arises in the basal telencephalon and contains morphologically homogenous neurons that can be divided into two distinct compartments, patches and the matrix. During development, patch neurons are generated first to form a striatal primordium. After a large influx of later-born matrix neurons into this region, the unique mosaic arrangement of these two neuronal phenotypes is established. The massive migration of matrix neurons continues during the embryonic period, and they eventually comprise 80-85% of the mature striatum. To elucidate the cellular mechanism or mechanisms underlying this critical event in striatal histogenesis, we examined the migration characteristics of striatal subventricular zone (SVZ) cells at embryonic day 18 when neurogenesis peaks for matrix neurons. Using gel cultures, we show that netrin-1, one of the diffusible guidance cues expressed in the striatal ventricular zone (VZ), exerts a repulsive action on migrating SVZ cells. This effect is blocked in the presence of antibodies against Deleted in colorectal cancer (DCC), a putative receptor for netrin-1. The expression patterns of netrin-1 and DCC strongly suggest the involvement of this effect in the outward migration of SVZ cells into the striatal postmitotic region. Our cell tracing study using living brain slices demonstrates that striatal SVZ cells migrate toward and disperse throughout the striatum, in which they differentiate into phenotypes of striatal projection neurons. We suggest that netrin-1 expressed in the striatal VZ serves to guide the large influx of striatal matrix neurons into the striatal primordium and is thereby involved in the initial formation of fundamental striatal structures.

The Slits are secreted proteins that bind to Robo receptors and play a role in axon guidance and neuronal migration. In vertebrates, Slit2 is a major chemorepellent for developing axons and is involved in the control of midline crossing. In vivo, Slit2 is cleaved into 140 kDa N-terminal (Slit2-N) and 55-60 kDa C-terminal (Slit2-C) fragments, although the uncleaved/full-length form can also be isolated from brain extract. We explored the functional activities of Slit2 fragments by engineering mutant and truncated versions of Slit2 representing the N-, C-, and full/uncleavable (Slit2-U) fragments. Only Slit2-N and Slit2-U bind the Robo proteins. We found that in collagen gel, olfactory bulb (OB) but not dorsal root ganglia (DRG) axons are repelled by Slit2-N and Slit2-U. Moreover, only Slit2-N membranes or purified protein-induced OB growth cones collapse. Finally, we found that only recombinant Slit2-N could induce branching of DRG axons and that this effect was antagonized by Slit2-U. Therefore, different axons have distinct responses to Slit2 fragments, and these proteins have different growth-promoting capacities.

Voltage- and frequency-dependent facilitation of calcium channel activity has been implicated in a number of key physiological processes. Various mechanisms have been proposed to mediate these regulations, including a switch between channel gating modes, voltage-dependent phosphorylation, and a voltage-dependent deinhibition of G-protein block. Studying such modulation on recombinant Ca channels expressed in oocytes, we previously reported that alpha(1C) L-type calcium channel contrast with non-L type Ca channels by its ability to exhibit facilitation by pre-depolarization (Voltage-dependent facilitation of a neuronal alpha(IC) L-type calcium channel, E. Bourinet et al., EMBO Journal, 1994; 13, 5032-5039). To further analyze this effect, we have investigated the molecular determinants which mediate the differences in voltage-dependent facilitation between "facilitable" alpha(1C) and "non facilitable" alpha(1E) calcium channels. We used a series of chimeras which combine the four transmembrane domains of the two channels. Results show that the four domains of alpha(1C) contribute to facilitation, with domain I being most critical. This domain is required but not sufficient alone to generate facilitation. The minimal requirement to observe the effect is the presence of domain I plus one of the three others. We conclude that similarly to activation gating, voltage-dependent facilitation of alpha(1C) is a complex process which involves multiple structural elements were domains I and III play the major role.

Huntington's disease (HD) is a progressive neurodegenerative disorder with no effective treatment. Geldanamycin is a benzoquinone ansamycin that binds to the heat shock protein Hsp90 and activates a heat shock response in mammalian cells. In this study, we show by using a filter retardation assay and immunofluorescence microscopy that treatment of mammalian cells with geldanamycin at nanomolar concentrations induces the expression of Hsp40, Hsp70 and Hsp90 and inhibits HD exon 1 protein aggregation in a dose-dependent manner. Similar results were obtained by overexpression of Hsp70 and Hsp40 in a separate cell culture model of HD. This is the first demonstration that huntingtin protein aggregation in cells can be suppressed by chemical compounds activating a specific heat shock response. These findings may provide the basis for the development of a novel pharmacotherapy for HD and related glutamine repeat disorders.

It has been proposed that elevated levels of tissue iron increase the risk for atherosclerosis, perhaps by favoring the formation of pro-atherogenic oxidized LDL. Working with apoE-deficient (apoE(-/-)) mice, which do not require a high-fat diet to develop atherosclerosis, we compared the effects of standard diet (0.02% iron) or a 2% carbonyl iron diet. After 24 weeks, mice fed the 2% carbonyl iron diet had twice as much iron in their plasma, a ninefold increase in bleomycin-detectable free iron in their plasma, and ten times as much iron in their livers as control mice. Dietary iron overload caused a modest (30%) rise in plasma triglyceride and cholesterol. Nevertheless, this regimen did not exacerbate, but rather reduced the severity of atherosclerosis by 50%, and it failed to elevate hepatic levels of heme oxygenase mRNA, which is induced by many different oxidative insults in vitro. Moreover, hepatic levels of protein-bound dityrosine and ortho-tyrosine, two markers of metal-catalyzed oxidative damage in vitro, failed to rise in iron-overloaded animals. Our observations suggest that elevated serum and tissue levels of iron are not atherogenic in apoE(-/-) mice. Moreover, they call into question the hypothesis that elevated levels of tissue iron promote LDL oxidation and oxidative stress in vivo.

Members of hereditary nonpolyposis colon cancer (HNPCC) families harboring heterozygous germline mutations in the DNA mismatch repair genes hMSH2 or hMLH1 present with tumors generally two to three decades earlier than individuals with nonfamilial sporadic colon cancer. We searched for phenotypic features that might predispose heterozygous cells from HNPCC kindreds to malignant transformation. hMSH2(+/-) lymphoblastoid cell lines were found to be on average about 4-fold more tolerant than wild-type cells to killing by the methylating agent temozolomide, a phenotype that is invariably linked with impairment of the mismatch repair system. This finding was associated with an average 2-fold decrease of the steady-state level of hMSH2 protein in hMSH2(+/-) cell lines. In contrast, hMLH1(+/-) heterozygous cells were indistinguishable from normal controls in these assays. Thus, despite the fact that HNPCC families harboring mutations in hMSH2 or hMLH1 cannot be distinguished clinically, the early stages of the carcinogenic process in hMSH2 and hMLH1 mutation carriers may be different. Should hMSH2(+/-) colonocytes and lymphoblasts harbor a similar phenotype, the increased tolerance of the former to DNA-damaging agents present in the human colon may play a key role in the initiation of the carcinogenic process.

<h4>Background and aims</h4>Hepatic steatosis has been shown to be associated with lipid peroxidation and hepatic fibrosis in a variety of liver diseases including non-alcoholic fatty liver disease. However, the lobular distribution of lipid peroxidation associated with hepatic steatosis, and the influence of hepatic iron stores on this are unknown. The aim of this study was to assess the distribution of lipid peroxidation in association with these factors, and the relationship of this to the fibrogenic cascade.<h4>Methods</h4>Liver biopsies from 39 patients with varying degrees of hepatic steatosis were assessed for evidence of lipid peroxidation (malondialdehyde adducts), hepatic iron, inflammation, fibrosis, hepatic stellate cell activation (alpha-smooth muscle actin and TGF-beta expression) and collagen type I synthesis (procollagen alpha1 (I) mRNA).<h4>Results</h4>Lipid peroxidation occurred in and adjacent to fat-laden hepatocytes and was maximal in acinar zone 3. Fibrosis was associated with steatosis (P < 0.04), lipid peroxidation (P < 0.05) and hepatic iron stores (P < 0.02). Multivariate logistic regression analysis confirmed the association between steatosis and lipid peroxidation within zone 3 hepatocytes (P < 0.05), while for hepatic iron, lipid peroxidation was seen within sinusoidal cells (P < 0.05), particularly in zone 1 (P < 0.02). Steatosis was also associated with acinar inflammation (P < 0.005). alpha-Smooth muscle actin expression was present in association with both lipid peroxidation and fibrosis. Although the effects of steatosis and iron on lipid peroxidation and fibrosis were additive, there was no evidence of a specific synergistic interaction between them.<h4>Conclusions</h4>These observations support a model where steatosis exerts an effect on fibrosis through lipid peroxidation, particularly in zone 3 hepatocytes.

Hereditary haemochromatosis is an inherited disorder characterised by an excessive iron absorption from the diet and is associated with several HFE gene mutations. One hypothesis is that these genetic mutations originated in the Celtic populations. The aim of this study is to determine the frequency of HFE gene mutations in a clustered Italian population of Celtic ancestry (Cimbri, Asiago plateau). One hundred and forty-nine consecutive unrelated blood donors (31 females and 118 males) were enrolled in this study. A family investigation was performed in each case to identify the ethnic origin of the individuals. The analysis of HFE gene mutations was performed by PCR amplification followed by digestion with RsaI and DpnII restriction enzymes. At least one HFE gene mutation was identified in 49 individuals (32.9%) of the studied population. The allele frequencies of the C282Y and H63D were respectively 0.037 and 0.144. When we considered only the 103 individuals with relatives born in Asiago, the prevalence of the HFE mutations rose from 32.9 to 39.8%; the allele frequencies of the C282Y and H63D were respectively 0.048 and 0.174. The mean serum iron and ferritin levels were significantly higher in individuals with the HFE mutations than in normal cases. This study indicates that the prevalence of the HFE gene mutations is surprisingly high in Italians with Celtic ancestry. This could suggest the need to perform large mass studies in selected areas of the country to detect the affected patients and prevent the disease in homozygous individuals.

The cause of Huntington's disease (HD) is a pathological expansion of the polyglutamine domain within the N-terminal region of huntingtin. Neuronal intranuclear inclusions and cytoplasmic aggregates composed of the mutant huntingtin within certain neuronal populations are a characteristic hallmark of HD. However, how the expanded polyglutamine repeats of mutant huntingtin cause HD is not known. Because in vitro expanded polyglutamine repeats are excellent glutaminyl-donor substrates of tissue transglutaminase (tTG), it has been hypothesized that tTG may contribute to the formation of these aggregates in HD. However, an association between huntingtin and tTG or modification of huntingtin by tTG has not been demonstrated in cells. To examine the interactions between tTG and huntingtin human neuroblastoma SH-SY5Y cells were stably transfected with full-length huntingtin containing 23 (FL-Q23) (wild type) or 82 (FL-Q82) (mutant) glutamine repeats or a truncated N-terminal huntingtin construct containing 23 (Q23) (wild type) or 62 (Q62) (mutant) glutamine repeats. Aggregates were rarely observed in the cells expressing full-length mutant huntingtin, and no specific colocalization of full-length huntingtin and tTG was observed. In contrast, in cells expressing truncated mutant huntingtin (Q62) there were numerous complexes of truncated mutant huntingtin and many of these complexes co-localized with tTG. However, the complexes were not insoluble structures. Further, truncated huntingtin coimmunoprecipitated with tTG, and this association increased when tTG was activated. Activation of tTG did not result in the modification of either truncated or full-length huntingtin, however proteins that were associated with truncated mutant huntingtin were selectively modified by tTG. This study is the first to demonstrate that tTG specifically interacts with a truncated form of huntingtin, and that activated tTG selectively modifies mutant huntingtin-associated proteins. These data suggest that proteolysis of full-length mutant huntingtin likely precedes its interaction with tTG and this process may facilitate the modification of huntingtin-associated proteins and thus contribute to the etiology of HD.

Huntington's disease (HD) and Dentatorubral and pallidoluysian atrophy (DRPLA) are autosomal dominant, neurodegenerative disorders caused by the expansion of polyglutamine tracts in their respective proteins, huntingtin and atrophin-1. We have previously generated mouse models of these disorders, using transgenes expressed via the prion protein promoter. Here, we report the first direct comparison of abnormalities in these models. The HD mice show abbreviated lifespans (4-6 months), hypoactivity, and mild impairment of motor skills. The DRPLA mice show severe tremors, are hyperactive, and are profoundly uncoordinated. Neuropathological analyses reveal that the distribution of diffuse nuclear immunolabeling and neuronal intranuclear inclusions (NII's), in the CNS of both models, was remarkably similar. Cytoplasmic aggregates of huntingtin were the major distinguishing neuropathological feature of the HD mice; mutant atrophin-1 accumulated/aggregated only in the nucleus. We suggest that the distinct behavioral and neuropathological phenotypes in these mice reflect differences in the way these mutant proteins perturb neuronal function.

Most patients with hereditary hemochromatosis are homozygous for C282Y in the HFE gene in populations of Celtic origin, but the genetic cause of this disease is unknown in Japan because of its rarity. A 48-year-old Japanese patient was recently diagnosed with idiopathic hemochromatosis. Analysis of the entire coding region of the patient's HFE by RT-PCR showed a heterozygous nucleotide substitution at nucleotide 527 from C to T, which resulted in A176V amino acid substitution. Another mutation at nucleotide 942 from T to C was observed, but this was a nonsense mutation. C282Y and another mutation, H63D, were not found in the patient. The mutation may have a possible role on the cause of hemochromatosis in this Japanese case.

The human major histocompatibility complex (MHC) on chromosome 6 encodes three classical class-I genes: human leukocyte antigens (HLA) A, B, and C. These polymorphic genes encode a 43- to 45-kDa cell surface glycoprotein that, in association with the 12-kDa beta2-microglobulin molecule, functions in the presentation of nine amino acid peptides to the T-cell receptor of CD8-bearing T lymphocytes and killer inhibitory receptors on natural killer cells. In addition to these ubiquitously expressed, polymorphic proteins, the human genome also encodes several nonclassical MHC class-I-like, or class Ib, genes that, in general, encode nonpolymorphic molecules involved in various specific immunological functions. Many of these genes, including CD1, the neonatal Fc receptor for IgG, HLA-G, HLA-E, the MHC class-I chain-related gene A, and Hfe, are prominently displayed on epithelial cells, suggesting an important role in epithelial cell biology.

To further examine the interrelationships between manganese and iron absorption, the mucosal uptake, initial rate of loss, whole-body retention, and tissue distribution of an orally administered 54Mn radiotracer were compared between normal and beta2-microglobulin knockout [beta2m(-/-)] mice. These mutant mice are commonly used as a model for the study of human hemochromatosis, a hereditary iron-overload disease. Initial uptake of 54Mn by the intestinal mucosa, the liver, and the brain was not different between the two strains. The mutant mice had much higher concentrations of nonheme and total iron in the liver, but hepatic manganese, copper, magnesium, and zinc concentrations were similar between the two strains. In summary, the mucosal uptake and whole-body retention of manganese and tissue manganese concentrations were not altered in beta2m(-/-) mice; this suggests that normal homeostasis of manganese is not affected by the altered HFE protein-beta2m complex in these mice.

Disturbances of the serotonergic pathway have been implicated in many psychiatric disorders, including alcoholism, aggression, schizophrenia and depression. The personality dimension of harm avoidance is correlated positively with the activity of mesolimbic serotonergic neurons. The goal of this study was to determine the role of the genes in this pathway in the development of type II alcoholism. A sample of alcoholics and normal controls were screened with the variations in tryptophan hydroxylase (TPH), serotonin receptors (5-HT2A and 5-HT2C), serotonin transporter (5-HTT), and monoamine oxidase A (MAO-A) genes. The results of association studies for type II alcoholics were the most significant with 5-HTT (P = 0.011) and MAO-A (P = 0.029) genes. However, after correction for multiple comparisons, none of the results reached the significance level. These data indicate that the genes in the serotonergic pathway may be involved in the development of type II alcoholism but the gene effects are very small.

A gene called deleted in colon cancer (DCC) has been identified on a region of chromosome 18, which is deleted in 70% of colorectal cancers. The DCC gene encodes a protein belonging to the immunoglobulin superfamily with similarity to the N-CAM transmembrane proteins, and it is a putative tumor-suppressor gene. Alternative splicing of transcripts of transmembrane proteins, including N-CAM, is know to occur, resulting in different isoforms of the protein. Using a polyclonal antibody against the DCC gene product, we have demonstrated, by antigen retrieval immunostaining, the presence of a DCC protein isoform on the cell surface of goblet cells in the G-I tract, cytoplasm of squamous epithelium in the skin & esophagus and transitional epithelium in the urinary bladder, ductal glandular epithelium of endometrium, endocervix, prostate, gall bladder and breast, cytoplasm of neuron in the cerebral cortex and Purkinje cells in the cerebellum. In addition, we also demonstrated DCC protein expression in neuroendocrine cells including argentaffin cells of the stomach, small intestine, appendix and colon, C cells of thyroid gland, chromaffin cells of the adrenal gland, islets of Langerhans in the pancreas and neurons of the sympathetic ganglion. This restrictive distribution suggests the DCC gene products may be abundant expression in neuroendocrine cells of human tissue.

<h4>Aim</h4>To study the genetic alteration in ACF and to define the possibility that ACF may be a very early morphological lesion with molecular changes,and to explore the relationship between ACF and colorectal adenoma even carcinoma.<h4>Methods</h4>DNA from 35 CRC, 15 adenomas, 34 ACF and 10 normal mucus was isolated by means of microdissection. Direct gene sequencing of K-ras gene including codon 12, 13 and 61 as well as the mutation cluster region (MCR) of APC gene was performed.<h4>Results</h4>K-ras gene mutation frequency in ACF, adenoma and carcinoma was 17.6% (6/34), 13.3% (2/15), and 14.3% (5/35) respectively, showing no difference (P > 0.05) in K-ras gene mutation among three pathologic procedures. The K-ras gene mutation in adenoma, carcinoma and 4 ACF restricted in codon 12 (GGT GAT), but the other 2 mutations from ACF located in codon 13 (GGC GAC). K-ras gene mutation was found more frequently in older patients and patients with polypoid cancer. No mutation in codon 61 was found in the three tissue types. Mutation rate of APC gene in adenoma and carcinoma was 22.9% (8/35) and 26.7% (4/15), which was higher than ACF (2.9%) (P <0.05). APC gene mutation in carcinoma was not correlated with age of patients, location, size and differentiation of tumor.<h4>Conclusion</h4>ACF might be a very early morphological lesion in the tumorogenesis of colorectal tumor. The morphological feature and gene mutation status was different in ACF and adenoma. ACF is possibly putative microadenoma that might be the precursor of adenoma. In addition, the development of a subgroup of colorectal carcinomas might undergo a way of normal epithelium ACF carcinomas .

<h4>Objective</h4>To evaluate the pedigree of genetic alterations during the tumorigenesis of intrahepatic cholangiocarcinoma (ICC) and their correlation with clinicopathological features by analysis of loss of heterozygosity (LOH) in 6 tumor suppressor genes (APC, MCC, DCC, OGG1, p53 and RB1) and point mutations in Ki-ras-2 oncogene.<h4>Methods</h4>Genomic DNA was isolated from paraffin-embedded slides of 22 surgically resected ICC cases by microdissection-based PCR amplification and agarose gel electrophoresis. Genetic alterations were analyzed by direct DNA sequencing.<h4>Results</h4>The total frequency of alterations in 7 genes studied was 86.4% (19/22). Based on the pattern of altered genes and their correlation with clinicopathological parameters, the genetic alterations were classified into two groups: Group I (9/19, 47.4%): alterations in APC, MCC, DCC and Ki-ras-2,); Group II (10/19, 52.6%): alterations in p53, OGG1 and RB1. The average age of patients in Group I (mean age, 57.2 years) was significantly younger than those in Group II (mean age, 69.1 years) (P < 0.05).<h4>Conclusions</h4>The occurrence and development of ICC was closely related with the accumulation and cooperation of multiple genetic alterations. The genetic alterations of APC, MCC, DCC and Ki-ras-2 may play crucial roles in the early stage of development of ICC, and the genetic alterations of p53, OGG1 and RB1 may play important roles in accelerating advanced progression of ICC. The detection of the pedigree of genetic alterations in ICC may provide useful information for evaluating the state of tumor progression and clinic prognosis.

Severe pulmonary tuberculosis (PTB) is sometimes complicated with deep venous thrombosis (DVT). We have examined the role of possible hemostat›c disturbance, which are predisposing factors for venous thrombosis in patients with PTB. Coagulation and platelet function tests have been studied in 40 patients with severe PTB and 40 healthy control volunteers before therapy and they were compared with 30th day results and controls. Analysis in patients with active PTB showed anemia, leucocytosis, thrombocytosis, elevation in plasma fibrinogen, factor VIII, plasminogen activator inhibitor 1 (PAI-1) with depressed antithrombin III (ATIII) and protein C (PC) levels. On the 30th day of treatment, anemia, leucocytosis and thrombocytsis were improved. Fibrinogen and factor VIII levels decreased to normal levels, PC and AT III levels increased to normal levels whereas there was no difference in PAI-1 levels. Platelet aggregation studies demonstrated increased platelet activation. Activated protein C resistance was not determined. DVT was not detected in patients during the follow up period. Decreased AT III, PC and elevated plasma fibrinogen levels and increased platelet aggregation appear to induce hypercoagilable state seen in PTB and improves with tretament.