Identification and mutation analysis of the complete gene for Chediak-Higashi syndrome.

Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive disorder characterized by hypopigmentation, severe immunologic deficiency with neutropenia and lack of natural killer (NK) cells, a bleeding tendency and neurologic abnormalities. Most patients die in childhood. The CHS hallmark is the occurrence of giant inclusion bodies and organelles in a variety of cell types, and protein sorting defects into these organelles. Similar abnormalities occur in the beige mouse, the proposed model for human CHS. Two groups have recently reported the identification of the beige gene, however the two cDNAs were not at all similar. Here we describe the sequence of a human cDNA homologous to mouse beige, identify pathologic mutations and clarify the discrepancies of the previous reports. Analysis of the CHS polypeptide demonstrates that its modular architecture is similar to the yeast vacuolar sorting protein, VPS15.

Identification of the murine beige gene by YAC complementation and positional cloning.

The beige mutation is a murine autosomal recessive disorder, resulting in hypopigmentation, bleeding and immune cell dysfunction. The gene defective in beige is thought to be a homologue of the gene for the human disorder Chediak-Higashi syndrome. We have identified the murine beige gene by in vitro complementation and positional cloning, and confirmed its identification by defining mutations in two independent mutant alleles. The sequence of the beige gene message shows strong nucleotide homology to multiple human ESTs, one or more of which may be associated with the Chediak-Higashi syndrome gene. The amino acid sequence of the Beige protein revealed a novel protein with significant amino acid homology to orphan proteins identified in Saccharomyces cerevisiae, Caenorhabditis elegans and humans.

Large improvements in major cardiovascular risk factors in the population of northern Sweden: the MONICA study 1986-2009.

OBJECTIVES: the incidence of cardiovascular disease has declined rapidly in Sweden since the 1980s. We explored changes in major cardiovascular risk factors in northern Sweden between 1986 and 2009. DESIGN: since 1986, six population surveys have been carried out in northern Sweden using procedures of the World Health Organization MONICA project. The population age range was 25-64 years in 1986 and 1990, and 25-74 years from 1994. Trends were analysed using generalized linear models. RESULTS: a total of 10586 subjects were included in the surveys. Blood pressure decreased by 4.9/3.9 mmHg in women and 1.8/1.5 mmHg in men aged 25-64 years between 1986 and 2009. In men and women aged 65-74 years, the decrease was 12.6/6.1 mmHg between 1994 and 2009. From 1994, the use of blood pressure-lowering drugs increased, particularly among the older subgroup. The prevalence of smoking halved between 1986 and 2009; 11% of women and 9% of men were smokers in 2009. Cholesterol levels decreased by 0.9 mmol L(-1) in the younger age group (25-64 years), and the use of lipid-lowering agents increased from 1994. Among subjects aged 25-64 years, one in five was obese in 2009, which was twice as many as in 1986, and body mass index (BMI) increased by 1.5 kg m(-2) , corresponding to an increase in weight of 4 kg. There was no further increase in BMI from 2004. The prevalence of diabetes did not change between 1986 and 2009. The proportion that received a university education increased markedly in all age groups, especially in women, during the study period. CONCLUSIONS: significant improvements were observed in major cardiovascular risk factors in northern Sweden between 1986 and 2009.

Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression.

In cancer patients, dormant micrometastases are often asymptomatic and clinically undetectable, for months or years, until relapse. We have studied dormant lung metastases under angiogenesis suppression in mice. The metastases exhibited rapid growth when the inhibition of angiogenesis was removed. Tumour cell proliferation, as measured by bromodeoxyuridine incorporation and immunohistochemical staining proliferating cell nuclear antigen, was not significantly different in dormant and growing metastases. However, tumour cells of dormant metastases exhibited a more than threefold higher incidence of apoptosis. These data show that metastases remain dormant when tumour cell proliferation is balanced by an equivalent rate of cell death and suggest that angiogenesis inhibitors control metastatic growth by indirectly increasing apoptosis in tumour cells.

Angiostatin induces and sustains dormancy of human primary tumors in mice.

There is now considerable direct evidence that tumor growth is angiogenesis-dependent. The most compelling evidence is based on the discovery of angiostatin, an angiogenesis inhibitor that selectively instructs endothelium to become refractory to angiogenic stimuli. Angiostatin, which specifically inhibits endothelial proliferation, induced dormancy of metastases defined by a balance of apoptosis and proliferation. We now show that systemic administration of human angiostatin potently inhibits the growth of three human and three murine primary carcinomas in mice. An almost complete inhibition of tumor growth was observed without detectable toxicity or resistance. The human carcinomas regressed to microscopic dormant foci in which tumor cell proliferation was balanced by apoptosis in the presence of blocked angiogenesis. This regression of primary tumors without toxicity has not been previously described. This is also the first demonstration of dormancy therapy, a novel anticancer strategy in which malignant tumors are regressed by prolonged blockade of angiogenesis.

Angiomotin: an angiostatin binding protein that regulates endothelial cell migration and tube formation.

Angiostatin, a circulating inhibitor of angiogenesis, was identified by its ability to maintain dormancy of established metastases in vivo. In vitro, angiostatin inhibits endothelial cell migration, proliferation, and tube formation, and induces apoptosis in a cell type-specific manner. We have used a construct encoding the kringle domains 1--4 of angiostatin to screen a placenta yeast two-hybrid cDNA library for angiostatin-binding peptides. Here we report the identification of angiomotin, a novel protein that mediates angiostatin inhibition of migration and tube formation of endothelial cells. In vivo, angiomotin is expressed in the endothelial cells of capillaries as well as larger vessels of the human placenta. Upon expression of angiomotin in HeLa cells, angiomotin bound and internalized fluorescein-labeled angiostatin. Transfected angiomotin as well as endogenous angiomotin protein were localized to the leading edge of migrating endothelial cells. Expression of angiomotin in endothelial cells resulted in increased cell migration, suggesting a stimulatory role of angiomotin in cell motility. However, treatment with angiostatin inhibited migration and tube formation in angiomotin-expressing cells but not in control cells. These findings indicate that angiostatin inhibits cell migration by interfering with angiomotin activity in endothelial cells.

Successive activation of the platelet-derived growth factor beta receptor and platelet-derived growth factor B genes correlates with the genesis of human choriocarcinoma.

The hydatidiform mole is a benign disease of the placenta characterized by the absence of the maternal genome. Approximately 3% of the reported cases will develop into malignant choriocarcinoma. In situ hybridization analysis reveals that the paternal platelet-derived growth factor (PDGF) beta receptor gene is up to 2 orders of magnitude more active in cytotrophoblasts of the complete hydatidiform moles than in normal placentae. The transition between hyperplasia (complete hydatidiform mole) and neoplasia (choriocarcinoma) in these cells correlates with at least a 10- to 20-fold activation of the PDGF-B gene. Since the neoplastic cytotrophoblasts have maintained an abnormally high level of PDGF beta receptor expression, we propose that a deregulated PDGF autostimulatory loop is involved in the genesis of human choriocarcinoma from hydatidiform moles.

Down-regulation of the novel gene melastatin correlates with potential for melanoma metastasis.

We have used differential cDNA display to search for genes whose expression correlates with an aggressive phenotype in variants of the B16 murine melanoma line, B16-F1 and B16-F10. This analysis identified a novel gene, termed melastatin, that is expressed at high levels in poorly metastatic variants of B16 melanoma and at much reduced levels in highly metastatic B16 variants. Melastatin was also found to be differentially expressed in tissue sections of human melanocytic neoplasms. Benign nevi express high levels of melastatin, whereas primary melanomas showed variable melastatin expression. Melastatin transcripts were not detected in melanoma metastases. Within the set of human primary cutaneous melanomas examined, melastatin expression appeared to correlate inversely with tumor thickness. The expression pattern observed suggests that loss of melastatin expression is an indicator of melanoma aggressiveness.

p53 induces angiogenesis-restricted dormancy in a mouse fibrosarcoma.

The p53 tumor-suppressor gene is inactivated in over 50% of all human cancers. In normal cells, p53 induces growth arrest and apoptosis in response to DNA damage. We show that p53 acts as potent tumor-suppressor gene independent of its well-documented effects on tumor-cell proliferation and apoptosis. p53 activates target genes in a murine fibrosarcoma cell-line but does not affect tumor cell-cycle progression or survival. Exogenous expression of wt-p53 does, however, block the angiogenic potential of the tumor cells resulting in formation of dormant tumors in vivo. These data provide evidence that: (1) p53 acts as a tumor suppressor gene independent of its anti-proliferative effects; (2) By inhibiting angiogenesis p53 can indirectly induce apoptosis in vivo but not in vitro; (3) p53-gene therapy which alters a tumors angiogenic potential, can revert tumors to a dormant phenotype.

Cellular localization of cytochrome(s) P-450 metabolizing polycyclic aromatic hydrocarbons in the rat adrenal cortex.

Cells were dispersed from the capsular, as well as the inner portion of female rat adrenal glands and subsequently separated on discontinuous Percoll gradients. The adrenal cells were distributed within a density interval ranging from 1.016 to 1.075 g/cm3 and different subpopulations showed distinct morphological appearances in suspension, as well as in culture. The total cells from the inner portion of the adrenals metabolized [14C]7,12-dimethylbenz(a)anthracene at a rate of 4.04 pmol/min 10(6) cells and synthesized corticosterone in response to ACTH stimulation at a rate of 1.07 micrograms/hr/10(6) cells. These activities were 4- and 2.5-fold higher, respectively, than the corresponding activities in cells isolated from the capsular portion. 7,12-Dimethylbenz(a)anthracene monoxygenase activity and ACTH-stimulated steroidogenesis were enriched in two subpopulations of cells obtained on the Percoll gradient and were estimated to be 13.1 pmol/min/10(6) cells and 3.21 micrograms/hr/10(6) cells, respectively, in the most active fraction (at the 1.034/1.040 g/cm3 interface). On the basis of cellular morphology, density and steroidogenic properties, it was concluded that adrenal 7,12-dimethylbenz(a)anthracene monoxygenase activity is localized mainly in the cells of the zona fasciculata.

Dose finding study of mosapride in functional dyspepsia: a placebo-controlled, randomized study.

BACKGROUND: Prokinetic agents have shown variable efficacy in the treatment of functional dyspepsia. Mosapride is a new prokinetic 5-hydroxytryptamine-4 agonistic agent. AIM: To evaluate the efficacy of three dosage regimens of mosapride compared with placebo in the treatment of functional dyspepsia. METHODS: Patients were randomly allocated to treatment with placebo or mosapride (5 mg b.d., 10 mg b.d. or 7.5 mg t.d.s.) in a double-blind, prospective, multicentre, multinational study. The change in symptom severity score from an untreated baseline week to the sixth week of treatment was used to compare treatment efficacy. RESULTS: There were 141, 140, 143 and 142 patients valid for evaluation in the intention-to-treat population in the placebo, mosapride 5 mg b.d., mosapride 10 mg b.d. and mosapride 7.5 mg t.d.s. groups, respectively. The mean changes in the overall dyspeptic symptom score were - 0.90, - 0.94, - 0.88 and - 0.89, respectively, and the proportions of patients feeling better at the end of the treatment period were 60%, 59%, 59% and 61%, respectively. No statistically significant difference was seen. CONCLUSIONS: Treatment of functional dyspepsia with mosapride was not superior to placebo. The result raises the question of whether treatment with prokinetic agents is appropriate for functional dyspepsia.

Horizontal transfer of tumor DNA to endothelial cells in vivo.

Tumor endothelial cells have long been regarded as genomically stable and therefore less likely to develop resistance to antiangiogenic therapies. However, recent findings have challenged this notion. We have shown that DNA can be transferred between cells through phagocytosis of apoptotic bodies by adjacent viable cells. Propagation of the ingested DNA is prevented by the activation of the p53-p21 pathway. In this study, we examined whether concomitant transfer of tumor DNA with genes that inactivate the p53 pathway could overcome the barrier to tumor DNA propagation. Our results demonstrate that fibroblasts and endothelial cells are capable of acquiring and replicating tumor DNA when the apoptotic tumor cells contain the SV40 large T antigen. Analysis of the tumor stroma of xenotransplanted tumors in severe combined immunodeficient mice revealed that a sub-population of the endothelial cells contained tumor DNA. These cells maintained the ability to form functional vessels in an in vivo assay and concurrently express tumor-encoded and endothelial-specific genes.

Sense of coherence in different stages of health and disease in northern Sweden--gender and psychosocial differences.

OBJECTIVE: To investigate "Sense of Coherence" (SOC) and its relation to perceived health, different stages of disease, and different psychosocial factors in a population-based study. DESIGN: Postal survey of a population-based sample, the MONICA study (1994). SETTING: Norrbotten and Västerbotten, the two northernmost counties in Sweden, with a total population of 510000 inhabitants. SUBJECTS: 837 men and 882 women in three mutually-exclusive groups: stomach trouble of many years' standing, identified disease (stroke, cardiac infarction, diabetes, anti-hypertension treatment) and no reported disease. MAIN OUTCOME MEASURES: SOC scores in relation to sociodemographic variables and perceived health. RESULTS: We found a relationship between low SOC scores and poor perceived health, low social support and low emotional support on a population level. When comparing persons with stomach trouble with those without disease, or with established diseases, we found similar relationships between low mean SOC scores in all strata for both women and men. "Perceived health", however, was only significantly correlated for women, and women had an overall stronger relationship. CONCLUSIONS: In a study in northern Sweden, female patients with stomach trouble comprise a vulnerable group. The concept of SOC introduces a new dimension for perceiving health and disease. In clinical practice, care providers can identify and elaborate on the relationship between SOC scores and sociodemographic data.

The cell type-specific IGF2 expression during early human development correlates to the pattern of overgrowth and neoplasia in the Beckwith-Wiedemann syndrome.

Overstimulation by insulin-like growth factor II is implied in several overgrowth conditions and childhood cancers. We have therefore studied spatial and temporal expression patterns of the insulin-like growth factor II gene (IGF2) and the insulin-like growth factor type 1 receptor gene during normal human development (5.5 to 23.0 weeks postfertilization). The set of cell types with the most abundant IGF2 expression correlated strikingly to the organomegaly and tumor predisposition of the Beckwith-Wiedemann syndrome. Intrauterine growth and postnatal organ weights of a prematurely born child with a full-blown syndrome are presented. The cell type-specific IGF2 expression of these organs and of multifocal Wilms' tumors from two other children affected by the Beckwith-Wiedemann syndrome were also studied. The results clarify and extend previous findings concerning human prenatal IGF2 expression and are consistent with a short range overstimulatory role of locally produced IGF II ensuing after the first trimester in the Beckwith-Wiedemann syndrome.

Angiogenesis during human extraembryonic development involves the spatiotemporal control of PDGF ligand and receptor gene expression.

We have examined the role of platelet-derived growth factor (PDGF) ligand and receptor genes in the angiogenic process of the developing human placenta. In situ hybridization analysis of first trimester placentae showed that most microcapillary endothelial cells coexpress the PDGF-B and PDGF beta-receptor genes. This observation indicates that PDGF-B may participate in placental angiogenesis by forming autostimulatory loops in capillary endothelial cells to promote cell proliferation. Endothelial cells of macro blood vessels maintained high PDGF-B expression, whereas PDGF beta-receptor mRNA was not detectable. In contrast, PDGF beta-receptor mRNA was readily detectable in fibroblast-like cells and smooth muscle cells in the surrounding intima of intermediate and macro blood vessels. Taken together, these data suggest that the PDGF-B signalling pathway appears to switch from an autocrine to a paracrine mechanism to stimulate growth of surrounding PDGF beta-receptor-positive mesenchymal stromal cells. Smooth muscle cells of the blood vessel intima also expressed the PDGF-A gene, the protein product of which is presumably targeted to the fibroblast-like cells of the mesenchymal stroma as these cells were the only ones expressing the PDGF alpha-receptor. PDGF-A expression was also detected in columnar cytotrophoblasts where it may have a potential role in stimulating mesenchymal cell growth at the base of the growing placental villi. We discuss the possibility that the regulation of the PDGF-B and beta-receptor gene expression might represent the potential targets for primary angiogenic factors.

Overlapping patterns of IGF2 and H19 expression during human development: biallelic IGF2 expression correlates with a lack of H19 expression.

The spatial patterns of IGF2 and H19 gene expression are strikingly similar during parts of human embryonic/fetal and early postnatal development. Notable exceptions were found with the ciliary anlage of the embryonic retina and the choroid plexus/leptomeninges, where transcripts from the IGF2 but not the H19 locus could be detected. Moreover, in contrast to the other tissue samples examined, the choroid plexus/leptomeninges expressed both parental IGF2 alleles. Whilst RNase protection analysis revealed a weak activity of the P1 promoter in the choroid plexus/leptomeninges, the P2, P3 and P4 promoters were all active wherever IGF2 was expressed. We discuss these observations with respect to a hypothesized coordinated control of the reciprocally imprinted and closely linked IGF2 and H19 loci.

The expression of PDGF alpha- and beta-receptors in subpopulations of PDGF-producing cells implicates autocrine stimulatory loops in the control of proliferation in cytotrophoblasts that have invaded the maternal endometrium.

In order to explain the high proliferative potential of human placental cytotrophoblasts, we have addressed the potential involvement of platelet-derived growth factor (PDGF) ligand and receptors. Although PDGF is usually described as a mitogen for cells of mesenchymal origin, we show in this report that extra-villous term placental cytotrophoblasts express the PDGF alpha- and beta-receptor genes, both in vivo and in vitro. In addition, cytotrophoblasts produce significant amounts of PDGF-B protein. By immunohistochemical analysis of receptor expression, we found that the PDGF alpha-receptors could be detected at the cell surface, while the PDGF beta-receptors were only detected intracellularly. In addition, double immunostaining analysis showed that the PDGF alpha- and beta-receptor molecules are expressed in different subpopulations of cytotrophoblasts. The addition of PDGF-AA and PDGF-BB homodimers to cytotrophoblast primary cultures induced a significant increase in DNA synthesis. We conclude, therefore, that PDGF is a growth factor for placental cytotrophoblasts and suggest that the growth of cytotrophoblasts can partly be explained by a PDGF autostimulatory loop, limited by the number of receptor-positive cytotrophoblasts.

Functional gene transfer of HIV DNA by an HIV receptor-independent mechanism.

HIV-1 enters target cells mainly via binding to CD4 and its coreceptors. The presence of HIV-1 in CD4- cells suggests, however, that there exist other mechanisms for viral entry. Here it is reported that HIV-1 DNA may be transferred from one cell to another by uptake of apoptotic bodies in a CD4-independent way. This was investigated by coculturing CD4-, chemokine receptor CCR5- and CXCR4- human fetal fibroblasts with apoptotic HIV-1-infected HuT78 cells or apoptotic PBMC isolated from HIV-1-infected patients. After 2 wk of coculture, fibroblasts contained HIV-1 DNA and expressed HIV-1 proteins p24 and gp120. Transfer of HIV-1 DNA was verified by coculturing fibroblasts with apoptotic bodies derived from cells infected with a defective HIV-1 virus. These cells contain one integrated copy of a reverse transcriptase (RT)-negative HIV-1 strain (8E5/LAV RT- cells) and consequently cannot produce free virus. Intracellular HIV-1 gag DNA was detected in both fibroblasts and dendritic cells after coculture with apoptotic 8E5/LAV RT- cells. Transfer of viral DNA after uptake of apoptotic bodies may explain HIV-1 infection of CD4- cells in vivo and furthermore may be relevant for Ag presentation.

Insulin-like growth factor 2 and short-range stimulatory loops in control of human placental growth.

Substructures of the first-trimester human placenta (within 3 months post-conception) display 'pseudo-malignant' properties. We show here, by in situ hybridization, that the insulin-like growth factor 2 (IGF-2) gene expression is particularly active in the cytotrophoblasts, which dominate these structures. Because the majority of placental IGF-2 mRNA is polysomal in extracts of first-trimester placenta, the spatial pattern of IGF-2 transcripts generally also defines the pattern of IGF-2 production. In primary trophoblast cultures, rendered quiescent by serum starvation. IGF-2 performs as a human embryonic growth factor by activating cell cycle entry/progression. Although both type 1 and 2 IGF receptor mRNAs can be found co-distributed with IGF-2 mRNA during placental development (supporting an autocrine role for IGF-2), these occasional patterns are confined to cytotrophoblasts with low proliferative potential. The reciprocity in ligand and receptor expression patterns are discussed in terms of rate-limiting steps in the involvement of IGF-2 in the proliferative phenotype of the early human placenta.