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A method for O- and S-palmitoylation of non-protected peptides has been developed. The peptides are treated with excess of palmitoyl chloride in 100% trifluoroacetic acid for 10 min at room temperature. The acidic conditions prevent acylation of amino groups, which is only significant after prolonged treatment (hours to days). The tripeptides Gly-Cys-Phe and Gly-Ser-Phe were converted into the respective S- and O-palmitoylated compounds, and the hydrophobic pulmonary surfactant protein-C model peptides, LRIPCCPVNLKRLLVVV [SP-C(1-17)] and FGIPSSPVLKRLLILLLLLLLILLLILGALLMGL [SP-C(Leu)] were converted into their respective S,S- and O,O-dipalmitoylated peptides. The reactions were virtually quantitative, and the palmitoylated peptides were isolated in about 75-80% yield after reversed-phase HPLC purification. CD spectroscopy showed that S, S-dipalmitoylation of SP-C(1-17) affects the peptide secondary structure (substantial increase in the alpha-helix content) in dodecylphosphocholine micelles.

<h4>Background & aims</h4>Hepatic iron overload has been reported in various metabolic conditions, including the insulin-resistance syndrome (IRS) and nonalcoholic steatohepatitis (NASH). The aim of this study was to show that such hepatic iron overload is part of a unique and unrecognized entity.<h4>Methods</h4>A total of 161 non-C282Y-homozygous patients with unexplained hepatic iron overload were included. We determined the age; sex; presence of IRS (1 or more of the following: body mass index of >25, diabetes, or hyperlipidemia); serum iron tests and liver iron concentration (LIC; reference value, <36 micromol/g); liver function test results; C282Y and H63D HFE mutations; and liver histological status.<h4>Results</h4>Patients were predominantly male and middle-aged. Most (94%) had IRS. Transferrin saturation was increased in 35% (median, 42%; range, 13%-94%). LIC ranged from 38 to 332 micromol/g (median, 90 micromol/g), and LIC/age ratio ranged from 0.5 to 4.8 (median, 1.8). Allelic frequencies of both HFE mutations were significantly increased compared with values in normal controls (C282Y, 20% vs. 9%; H63D, 30% vs. 17%), only because of a higher prevalence of compound heterozygotes. Patients with no HFE mutations had similar degrees of iron overload as those with other genotypes, except for compound heterozygotes, who had slightly more iron burden. Steatosis was present in 25% of patients and NASH in 27%. Portal fibrosis (grades 0-3) was present in 62% of patients (grade 2 or 3 in 12%) in association with steatosis, inflammation, and increased age. Sex ratio, IRS, transferrin saturation, and LIC did not vary with liver damage. Serum ferritin concentration, liver function test results, and fibrosis grade were more elevated in patients with steatosis and NASH than in others, but LIC and allelic frequencies of HFE mutations were similar.<h4>Conclusions</h4>This study shows that patients with unexplained hepatic iron overload are characterized by a mild to moderate iron burden and the nearly constant association of an IRS irrespective of liver damage.

<h4>Objective</h4>To review the current state-of-the-art regarding the role of iron- and DNA-based testing on the detection, treatment, and prevention of hereditary hemochromatosis (HH), the most common single-gene disorder in white people.<h4>Sources</h4>Review of the medical literature, with particular emphasis on recent reports of the impact of DNA-based testing on the detection of symptomatic and presymptomatic patients with HH.<h4>Conclusions</h4>Hereditary hemochromatosis, a common autosomal recessive iron overload disorder (with a population prevalence of 0.3%-0.8%), is a common cause of preventable liver, heart, joint, and endocrine disease. Since the associated clinical signs and symptoms are nonspecific, an accurate HH diagnosis demands both a high index of suspicion and the direct laboratory demonstration of elevated iron parameters. The substantial public health burden of HH as a common, deadly, detectable, and treatable chronic disease has led the College of American Pathologists to recommend that "systematic screening for hemochromatosis is warranted for all persons over the age of 20 years." The recent discovery that most HH cases are the result of a single well-conserved homozygous missense mutation (C282Y) within a novel transferrin-receptor binding protein (HFE) has given rise to diagnostic clinical tests for the DNA-based detection of this pathologic mutation. This direct HFE mutation test can now be used not only to confirm the diagnosis of HH in those with symptomatic disease, but also, perhaps more importantly, to detect those with presymptomatic iron overload in whom future disease manifestations may be prevented (with phlebotomy therapy).

<h4>Background</h4>Changes in platelet aggregation (PA) and platelet surface receptor expression induced by thrombolytic therapy for acute myocardial infarction may influence the rate of initial reperfusion and early reocclusion.<h4>Methods and results</h4>In the RAPID-1 (Reteplase Angiographic Phase II International Dose-finding study), RAPID-2 (Reteplase vs Alteplase Patency Investigation During myocardial infarction), INJECT (INternational Joint Efficacy Comparison of Thrombolytics), and GUSTO-3 (Global Use of Strategies To Open occluded coronary arteries) trials, 126 patients were enrolled in a single center. Patients were treated with either conventional alteplase (100 mg/180 min; n=15), accelerated alteplase (100 mg/90 min; n=21), reteplase 10+10-U double bolus (n=50), reteplase 10+5-U double bolus (n=15), reteplase 15-U single bolus (n=15), or streptokinase (1.5 MU/60 min; n=10). PA (after stimulation with ADP), P-selectin expression and fibrinogen binding to glycoprotein (GP) IIb/IIIa (determined by flow cytometry with and without stimulation with ADP), and levels of soluble P-selectin, prothrombin fragments F1 and F2, thrombin-antithrombin complexes (TAT), and antithrombin III (ATIII) were determined. PA decreased significantly at 1 and 2 hours in patients treated by 10+10-U reteplase or by streptokinase. Fibrinogen binding to platelet GP IIb/IIIa followed a similar pattern. Significant thrombin generation and significantly elevated thrombin levels during thrombolysis were reflected by increased F1 and F2 fragments and TAT levels in all treatment groups. ATIII levels decreased significantly during thrombolytic therapy.<h4>Conclusions</h4>A decrease in PA in patients treated by reteplase or streptokinase compared with alteplase could be observed in the early phase. Double bolus (10+10 U) reteplase and streptokinase resulted in lower PA at 1 and 2 hours than therapy with accelerated alteplase. Total fibrinogen and fibrinogen binding to GP IIb/IIIa tended to be lower during the first 2 hours after reteplase than after accelerated alteplase.

<h4>Objective</h4>To analyze the genotypes of the promoter region of the serotonin transporter gene (5-HTT) in patients with fibromyalgia (FM).<h4>Methods</h4>Genomic DNA from 62 patients meeting the American College of Rheumatology 1990 criteria for FM and 110 healthy controls was analyzed by polymerase chain reaction. Additionally, the psychopathologic state of 52 of the FM patients was evaluated using the Beck Depression Inventory (BDI) and the Symptom Checklist-90-Revised (SCL-90-R).<h4>Results</h4>The 5-HTTLPR genotypes in FM patients versus controls were distributed as follows: L/L 27% versus 34%, L/S 42% versus 50%, and S/S 31% versus 16%. FM patients with the S/S genotype had higher mean scores on the BDI and the SCL-90-R compared with those in the L/L and L/S groups.<h4>Conclusion</h4>A higher frequency of the S/S genotype of 5-HTT was found in FM patients compared with healthy controls. The S/S subgroup exhibited higher mean levels of depression and psychological distress. These results support the notion of altered serotonin metabolism in at least a subgroup of patients with FM.

The I1307K polymorphism in APC has been found to predispose to colorectal cancer in Ashkenazi Jews, and has recently been associated with an increased risk for breast cancer in the same population. In that study, we genotyped 205 paraffin-embedded breast cancers from Ashkenazi Jewish women diagnosed below the age of 65. We now present an extended analysis, with clinicopathological correlations between carriers of I1307K and non-carriers. Twenty-four of 209 cases (11.5%, 95% confidence interval 7.5-16.6) were found to carry the I1307K polymorphism. When stratifying the data by other relevant clinicopathological variables, we observed no association between the presence of this polymorphism and age at diagnosis (P = 0.52), grade (P = 0.074), tumour size (P = 0.99), lymph node status (P = 0.82), oestrogen receptor status (P = 0.23) or P53 immunoreactivity (P = 0.80). The breast-cancer specific 5-year survival for women with I1307 K polymorphism was 88.9% compared with 81.6% in women without I1307K (P = 0.34). Using microdissected samples and direct sequencing, no somatic mutations were observed in any of the 24 I1307K-positive cases. Single-strand conformation analysis of 158 of the I1307K-negative breast cancers that were available for study revealed no mobility shifts. We conclude that the presence of the I1307K polymorphism does not appear to be associated with any particular clinicopathological feature of breast cancer and importantly, does not affect the prognosis.

HFE is a class I major histocompatibility complex (MHC)-related protein that is mutated in patients with the iron overload disease hereditary hemochromatosis. HFE binds to transferrin receptor (TfR), the receptor used by cells to obtain iron in the form of diferric transferrin (Fe-Tf). Previous studies demonstrated that HFE and Fe-Tf can bind simultaneously to TfR to form a ternary complex, and that membrane-bound or soluble HFE binding to cell surface TfR results in a reduction in the affinity of TfR for Fe-Tf. We studied the inhibition by soluble HFE of the interaction between soluble TfR and Fe-Tf using radioactivity-based and biosensor-based assays. The results demonstrate that HFE inhibits the TfR:Fe-Tf interaction by binding at or near the Fe-Tf binding site on TfR, and that the Fe-Tf:TfR:HFE ternary complex consists of one Fe-Tf and one HFE bound to a TfR homodimer.

The puzzling linkage between genetic hemochromatosis and histocompatibility loci became even more so when the gene involved, HFE, was identified. Indeed, within the well defined, mainly peptide-binding, MHC class I family of molecules, HFE seems to perform an unusual yet essential function. As yet, our understanding of HFE function in iron homeostasis is only partial; an even more open question is its possible role in the immune system. To advance on both of these avenues, we report the deletion of HFE alpha1 and alpha2 putative ligand binding domains in vivo. HFE-deficient animals were analyzed for a comprehensive set of metabolic and immune parameters. Faithfully mimicking human hemochromatosis, mice homozygous for this deletion develop iron overload, characterized by a higher plasma iron content and a raised transferrin saturation as well as an elevated hepatic iron load. The primary defect could, indeed, be traced to an augmented duodenal iron absorption. In parallel, measurement of the gut mucosal iron content as well as iron regulatory proteins allows a more informed evaluation of various hypotheses regarding the precise role of HFE in iron homeostasis. Finally, an extensive phenotyping of primary and secondary lymphoid organs including the gut provides no compelling evidence for an obvious immune-linked function for HFE.

In spite of marked changes in the glycosylation upon malignant transformation of cells, no biological significance of beta-1, 4-galactosyltransferase (beta-1,4-GalT) activities has been elucidated. When beta-1,4-GalT activities toward 1 mM GlcNAcbeta-S-pNP were determined using homogenates of NIH3T3 and its transformant, MTAg, MTAg contained 1.3 times higher activities. Northern blot analysis, however, revealed that the beta-1,4-GalT V gene expression increases by three times with a decrease in that of beta-1,4-GalT II by one-fifth and without significant changes in those of other beta-1,4-GalTs in MTAg. Analysis of beta-1,4-GalT V acceptor-specificity showed that the GlcNAcbeta1-->6Man group of the GlcNAcbeta1-->6(GlcNAbeta1-->2)Manalpha1- branch is galactosylated. These results indicate that changes in beta-1,4-GalT II and V activities are important for the altered glycosylation.

Optic nerve formation in mouse involves interactions between netrin-1 at the optic disk and the netrin-1 receptor DCC (deleted in colorectal cancer) expressed on retinal ganglion cell (RGC) axons. Deficiency in either protein causes RGC pathfinding defects at the disk leading to optic nerve hypoplasia (). Here we show that further along the visual pathway, RGC axons in netrin-1- or DCC-deficient mice grow in unusually angular trajectories within the ventral hypothalamus. In heterozygous Sey(neu) mice that also have a small optic nerve, RGC axon trajectories appear normal, indicating that the altered RGC axon trajectories in netrin-1 and DCC mutants are not secondarily caused by optic nerve hypoplasia. Intrinsic hypothalamic patterning is also affected in netrin-1 and DCC mutants, including a severe reduction in the posterior axon projections of gonadotropin-releasing hormone neurons. In addition to axon pathway defects, antidiuretic hormone and oxytocin neurons are found ectopically in the ventromedial hypothalamus, apparently no longer confined to the supraoptic nucleus in mutants. In summary, netrin-1 and DCC, presumably via direct interactions, govern both axon pathway formation and neuronal position during hypothalamic development, and loss of netrin-1 or DCC function affects both visual and neuroendocrine systems. Netrin protein localization also indicates that unlike in more caudal CNS, guidance about the hypothalamic ventral midline does not require midline expression of netrin.

<h4>Objective</h4>To investigate possible associations between serotonin transporter (5-HTT) promoter genotypic variants (l/l, l/s, and s/s) and differential regulation of platelet 5-HTT functionality parameters in a group of drug-naive depressed children and adolescents and healthy controls.<h4>Method</h4>Children and adolescents with major depression (n = 18) defined by DSM-III-R criteria and normal controls (n = 21) were assessed both for platelet serotonin functionality and for genotypic variants on 5-HTT promoter region. Four parameters were considered: (1) serotonin uptake rate (Vmax); (2) serotonin dissociation constant (K(m)); (3) paroxetine binding and density of site (Bmax); and (4) paroxetine dissociation constant (Kd).<h4>Results</h4>Depressed children had lower Vmax and K(m). Control subjects with l/l genotype had significantly higher Vmax than control subjects with l/s and s/s genotype. Control subjects with l/l genotype also had significantly higher Vmax than their depressed homologs. In contrast, Vmax was not significantly different between depressed and nondepressed subjects who carried the other 2 genotypes. The 5-HTT promoter genotype, diagnoses, or their interaction had no effect on the other serotonin parameters.<h4>Conclusions</h4>While showing a significant decrease of Vmax and K(m) in a group of drug-naive depressed children and adolescents, these data suggest that l/l genotype has a substantial effect on the decrease of Vmax during a depressive episode.

Erythroid 5-aminolevulinate synthase (ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells. Mutation of human ALAS-E causes the disorder X-linked sideroblastic anemia. To examine the roles of heme during hematopoiesis, we disrupted the mouse ALAS-E gene. ALAS-E-null embryos showed no hemoglobinized cells and died by embryonic day 11.5, indicating that ALAS-E is the principal isozyme contributing to erythroid heme biosynthesis. In the ALAS-E-null mutant embryos, erythroid differentiation was arrested, and an abnormal hematopoietic cell fraction emerged that accumulated a large amount of iron diffusely in the cytoplasm. In contrast, we found typical ring sideroblasts that accumulated iron mostly in mitochondria in adult mice chimeric for ALAS-E-null mutant cells, indicating that the mode of iron accumulation caused by the lack of ALAS-E is different in primitive and definitive erythroid cells. These results demonstrate that ALAS-E, and hence heme supply, is necessary for differentiation and iron metabolism of erythroid cells.

Large-scale sequencing projects have predicted high numbers of gene products for which no functional information is yet available. Hence, large-scale projects, such as gene knockouts, gene expression profiles, and protein-interaction mapping, are currently under way to initiate the understanding of the function of these gene products. The high-throughput strategies that are currently being developed to generate protein-interaction maps include automated versions of the yeast two-hybrid system. These strategies rely on the large-scale construction of DNA-binding domain/protein-of-interest hybrid constructs (DB-X baits). An inherent problem of large-scale two-hybrid systems is that a high percentage of cloned sequences encode polypeptides that, when fused to DB, can activate transcription in the absence of any two-hybrid-interacting partner protein. Here, we describe and validate a genetic strategy that efficiently eliminates such self-activator baits prior to screening procedures. The strategy is based on a negative-growth selection and is compatible with high-throughput settings.

Class I MHC protein primarily presents endogenous antigen but also may present exogenous antigen. Here, we investigated the intracellular pathway of spontaneously internalized class I MHC protein by confocal microscopy. beta(2)-microglobulin (beta(2)m), labeled with a single fluorophore, was exchanged at the surface of B cell transfectants to specifically mark cell surface and endocytosed class I MHC protein. Intracellular beta(2)m colocalized with fluorophore-conjugated transferrin, implying that class I MHC protein endocytosed into early endosomes. These endosomes containing fluorescent beta(2)m were found close to or within the Golgi apparatus, marked by fluorescent ceramide. Even after 24 hr of incubation, very little fluorescent beta(2)m was found in intracellular organelles stained by DiOC(6), marking the endoplasmic reticulum, or fluorophore-conjugated low density lipoprotein, marking late endosomes and lysosomes. Fluorophore-conjugated superantigens (staphylococcal enterotoxin A and B), presumed to enter cells bound to class II MHC protein, also were found to endocytose into beta(2)m-containing early endosomes. Staining with mAb and use of transfectants expressing MHC protein attached to green fluorescent protein confirmed the presence of intracellular compartments rich in both class I and II MHC protein and demonstrated that class I and II MHC protein also colocalize in discrete microdomains at the cell surface. These cell surface microdomains also contained transferrin receptor and often were juxtaposed to cholesterol-rich lipid rafts. Thus, class I and II MHC protein meet in microdomains of the plasma membrane and endocytose into early endosomes, where both may acquire and present exogenous antigen.

The yeast prion-like protein Sup35 has repeats responsible for the reversible induction of an altered, but advantageous phenotype. The expansion of similar repeat domains in several mammalian proteins is associated with neurodegenerative disease - so why are these 'bungee cord' domains conserved?

Various colon carcinoma cell lines were tested in different invasion assays, i.e. invasion into Matrigel, into confluent fibroblast layers and into chicken heart tissue. Furthermore, invasive capacity and metastatic potential were determined in nude mice. The colon carcinoma cells used were the human cell lines Caco-2, SW-480, SW-620 and HT-29, and the murine lines Colon-26 and -38. None of the human colon carcinoma cells migrated through porous membranes coated with Matrigel; of the murine lines, only Colon-26 did. When incubated in a mixture of Matrigel and culture medium non-invading cells formed spheroid cultures, whereas invading cells showed a stellate outgrowth. Only the heterogeneously shaped (epithelioid and stellate) cells of SW-480 and SW-620 and the spindle-shaped cells of Colon-26 invaded clearly confluent skin and colon fibroblasts as well as chicken heart tissue. However, when transplanted into the caecum of nude and syngeneic mice, all the lines tested were invasive with the exception of Caco-2 cells. We conclude that the outcome of in vitro tests measuring the invasive capacity of neoplastic cells is largely dependent on the test system used. Invasive capacity in vitro is strongly correlated with cells having a spindle cell shape, vimentin expression and E-cadherin down regulation. In contrast, HT-29 and Colon-38 cells having an epithelioid phenotype were clearly invasive and metastatic in vivo, but not in vitro.

We report a rare case of Crohn disease accompanied by a small-bowel carcinoma that developed in a 54-year-old Japanese man. The ulcerating tumor, which histologically proved to be a poorly differentiated adenocarcinoma and dysplasia surrounding the carcinoma, was located in the diseased ileum. The Ki-67 immunoreactive epithelial cells were increased in regenerative mucosa as compared with values for normal mucosa. The Ki-67- and p53-positive cells were increased in dysplasia and carcinoma as compared with values for regenerative or normal mucosa. In contrast, the p21(WAF1/CIP1) immunoreactive cells were decreased in this order. Intense DCC (deleted in colorectal cancer) expression was constantly shown among normal, regenerative, dysplastic and cancerous tissues. No bcl-2 expression and c-Ki-ras mutations were apparent. In conclusion, enhanced epithelial cell proliferation, p53 overexpression, and decrease of p21(WAF1/CIP1) expression may predispose the small-bowel mucosa to dysplasia and carcinoma development in Crohn disease.

Hereditary hemochromatosis is an autosomal, recessive disorder of the iron metabolism. The hemochromatosis gene (HFE) was previously located on chromosome 6 and recently identified by positional cloning. A point mutation, C282Y, was found to be present in the HFE gene in homozygous form in 64 to 100% of patients with established hemochromatosis. The relationship of a second polymorphic variant of the HFE gene, H63D to the formation of iron overload is debated. Although hemochromatosis is one of the most common inherited disorders among Caucasians, in the absence of specific signs it is rarely diagnosed. In order to obtain comparable epidemiological data for Hungary, we tested 1271 and 277 randomly selected, unrelated, healthy subjects for C282Y and H63D respectively. In addition C282Y testing was carried out in 58 patients suffering from liver cirrhosis, and in 191 individuals with suspected hemochromatosis. For C282Y and H63D mutation analyses polymerase chain reaction technique followed by Rsa I and Bcl I restriction enzyme digestion was used. We developed an alternative method for the detection of C282Y based on an amplification-generated Kpn I restriction site. The allele frequencies were 3.8% and 12.3% for C282Y and H63D respectively in the normal Hungarian population. There was no significant difference in C282Y allele frequencies between liver disease patients (1.7%) and the normal population. We identified 15 homozygous and 25 heterozygous individuals among 191 individuals with suspected hemochromatosis. The C282Y and the H63D allele frequencies in the normal Hungarian population were found to be similar to the allele frequencies observed in other European populations, indicating that there is a large number of individuals susceptible for iron overload in Hungary (1:700). Mutation analysis is a novel, non-invasive method in the diagnostics of hereditary hemochromatosis, which increasingly becomes part of the routine clinical work.

<h4>Background and purpose</h4>An outbreak of enterovirus infection occurred in Taiwan from late spring to early fall of 1998. Most of the pediatric infections presented as hand-foot-mouth disease (HFMD) and herpangina. A small portion of patients had symptoms of polio-like encephalitis and paralysis. The purpose of this study was to review the MR imaging findings in CNS involvement of enterovirus infection.<h4>Methods</h4>Twenty patients who had HFMD and clinical encephalitis were examined with MR imaging. T1-weighted and T2-weighted MR images were obtained. From the rectum, throat, CSF, and peripheral blood, the presence of enterovirus 71 (EV 71) was determined by virus culture, immunofluorescent microscopy, immunologic dot blotting, and reverse-transcription polymerase chain reaction.<h4>Results</h4>MR imaging studies of 20 patients showed hyperintensity in the brain stem and spinal cord in 15 patients, as seen on T2-weighted images. The major CNS lesions were in the medulla oblongata, pons, midbrain, and the dentate nuclei of the cerebellum. In some cases, the lesions involved the spinal cord (three cases) as well as the thalamus (two cases) and putamina (one case). Five patients had normal MR imaging results. After the appropriate management for tachycardia and tachypnea, 18 patients recovered within 1 to 2 weeks. In the follow-up MR imaging examination of five patients, the lesions completely disappeared within 2 weeks to 2 months. In two patients who were still respirator-dependent, MR imaging showed the tissue destruction in the posterior portions of the medulla, pons, and the ventral horns of cervical spinal cord. In one patient, most of midbrain was damaged. The presence of EV 71 was detected in specimens from 18 patients.<h4>Conclusion</h4>Because EV 71 was identified in 18 patients, and no other virus was detected, EV 71 was determined to be the major causative agent of this encephalomyelitis. Brain stem and cervical spinal cord involvement are characteristic findings of enteroviral encephalomyelitis.

Linker histones (e.g. H1, H5, H1 degrees ) are thought to exert control on chromatin function by restricting nucleosomal dynamics. All higher eukaryotes possess a diverse family of linker histones, which may exhibit functional specialization. Arabidopsis thaliana apparently contains a minimal complement of linker histone structural variants and therefore is an ideal model for investigating functional differentiation among linker histones. Histones H1-1 and H1-2 are relatively similar proteins that are expressed in a wide variety of tissues and make up the majority of linker histone while H1-3 is a highly divergent minor variant protein that is induced by drought stress. We are interested in determining whether the in vivo distribution of each of these proteins also differs. To this end, we have produced subtype-specific antibodies and have localized each of the three proteins at the intranuclear and DNA sequence levels by indirect immunofluorescence and immunoprecipitation, respectively. Antibodies against linker histones H1-1 and H1-2 decorate nuclei in patterns very similar to 4',6-diamidino-2-phenylindole (DAPI) staining, but different than the staining pattern of total histones. In contrast, antibodies made against two regions of H1-3 bind to chromatin in a diffuse pattern distinct from the DAPI-staining pattern. We also describe a technique to determine the localization of plant linker histone variants along regions of chromatin, employing in vivo chemical DNA-protein cross-linking to preserve native associations followed by immunoprecipitation with subtype-specific antibodies. We use this technique to demonstrate that, in contrast to the major linker histones, H1-3 does not bind the repetitive sequences pAL1 and 5S rDNA. In addition, we show that linker histones are bound to the compacted nucleosomal arrays at the telomere but with reduced stoichiometry. Taken together, our results suggest that plants, as has been shown for animals, possess a variant linker histone that is differentially localized.

Long distance cell migration occurs throughout the developing CNS, but the underlying cellular and molecular mechanisms are poorly understood. We show that the directed circumferential migration of basilar pontine neurons from their origin in the neuroepithelium of the dorsal hindbrain to the ventral midline involves the extension of long (>1 mm) leading processes, which marker analyses suggest are molecularly distinct from axons. In vivo analysis of knockout mice implicates the axonal chemoattractant netrin-1, functioning via its receptor Deleted in Colorectal Cancer (DCC), in attracting the leading process to the ventral midline. Direct evidence for this chemoattractant mechanism is provided, using explant cultures and time-lapse analysis in vitro. Our results demonstrate the attraction of migrating neurons in the mammalian brain by an axon guidance molecule and the chemotactic responsiveness of their leading processes.

Identification of the HFE gene and its C282Y and H63D mutations has improved the classification of iron overload disorders. Inherited hemochromatosis is due mainly, or perhaps only, to C282Y homozygosity, whereas nonhemochromatosis forms of iron overload are due to other HFE mutations and are usually responsible for mild overload precipitated by another factor such as cirrhosis or insulin-resistance. In practice, the diagnosis of inherited hemochromatosis rests on demonstration of homozygosity for the C282Y mutation; in this setting, the role of liver biopsy is to evaluate the prognosis by looking for fibrosis. In patients who are not homozygous for the C282Y mutation but have severe iron overload, causes other than hemochromatosis should be looked for before the extremely remote possibility of nonC282Y-related hemochromatosis is considered; here, liver biopsy remains of considerable diagnostic usefulness.

Bis(picolinato)oxovanadium(IV) [VO(pic)2] is one of the most potent insulin-mimetic vanadium complexes. To probe coordination structural changes of this complex in vivo and provide insights into the origin of its high potency, an electron spin-echo envelope modulation (ESEEM) study was performed on organs (kidney, liver and bone) of VO(pic)2- and VOSO4-treated rats. Kidney and liver samples from both types of rats exhibited a 14N ESEEM signal that could be attributed to equatorially coordinating amine nitrogen. The relative intensity of the amine signal was larger for the organs of the rat treated with the less potent VOSO4, suggesting that this amine coordination inhibits the insulin-mimetic activity. The spectra of kidney and liver from the VO(pic)2-treated rat contained a weak signal due to the picolinate imine nitrogen. This suggests that some picolinato species (including both the bispicolinato and a partially decomposed monopicolinato species) still exist in the organs as a minor species, where the proportions of the picolinato species to the total amount of the EPR-detectable VIVO species are estimated as 8-16% in the kidney and 12-24% in the liver. The picolinate ligand presumably serves to prevent VO2+ from being converted into the inactive amine-coordinated species. Bone samples from both types of rats exhibited an ESEEM signal due to 31P nuclei. The VO2+ in bone is therefore most likely incorporated into the hydroxyapatite Ca10(PO4)6(OH)2 matrix, which is consistent with the hypothesis that the bone-accumulated VO2+ is gradually released and transported to other organs as is Ca2+. No 14N signals were observed, even in the bone samples of the VO(pic)2-treated rats, indicating that vanadium uptake by bone requires complete decomposition of the complex.

Increasing knowledge of the molecular basis of disease and advances in technology for analyzing nucleic acids and gene products are changing pathology practice. The explosion of information regarding inherited susceptibility to disease is an important aspect of this transformation. Pathology residency programs are incorporating molecular pathology education into their curricula to prepare newly trained pathologists for the future, yet little guidance has been available regarding the important components of molecular pathology training. We present general goals for pathology training programs for molecular pathology education. These include recommendations to pathology residents for the acquisition of both basic knowledge in human genetics and molecular biology and specific skills relevant to microbiology, molecular oncology, genetics, histocompatibility, and identity determination. The importance of residents gaining facility in integrating data gained via nucleic acid based-technology with other laboratory and clinical information available in the care of patients is emphasized.

<h4>Objective</h4>To study the incidence, cause and clinical manifestation of venous thromboembolism during pregnancy and puerperium, and its diagnosis and treatment.<h4>Methods</h4>12 cases of venous thromboembolism admitted in our hospital from 1984-1997 were analysed retrospectively. The plasma protein C, protein S and antithrombin III activities were measured in 4 of the cases and activated protein C resistance (APC-R) were assayed by activated partial thrombinplastin time (APIT) in the presence and absence of APC (APC-APIT) and FV Leiden gene mutation were analysed by PCR restriction fragment length polymorphisms (RFLP) methods as well.<h4>Results</h4>Four cases occurred before delivery and 8 postpartum. Two cases complicated by pulmonary thromboembolism, and of them 1 died. APC-R(+), reduced activity of antithrombin-III and protein S each were found in 3 separate cases. No FV Leiden gene mutation was found in the 4 cases.<h4>Conclusions</h4>The formation of venous thromboembolism during pregnancy and puerperium is highly associated with the deficiency of anticoagulant proteins. Anticoagulation is recommended in the high risk women of thromboembolism.