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The molecular mechanisms and genetic changes that lead to the progression of prostate cancer during endocrine therapy are poorly characterized. Here, paired specimens from both untreated primary tumors and from local recurrences were collected from 10 prostate cancer patients treated by conventional androgen deprivation therapy. The genetic progression of the tumors was studied by using interphase fluorescence in situ hybridization and chromosome-specific probes. Six primary tumors (60%) and all ten recurrent tumors were aneuploid by fluorescence in situ hybridization. The recurrent tumors also showed a high degree of chromosome copy number variability from one cell to another. Increased copy number of chromosome X was particularly common in the recurrent tumors. In addition, specific high level amplification of the androgen receptor (AR) gene (Xq12) was detected in three highly aneuploid recurrent tumors. Our findings suggest that hormone-refractory prostate cancers are genetically very complex and show intratumor genetic heterogeneity. Increased copy number of chromosome X and the amplification of the androgen receptor (AR) gene may confer proliferative advantage during androgen deprivation and thus contribute to the development of recurrence.

<h4>Purpose</h4>Iron overload unexplained by dietary or medicinal iron excess, transfusion, or sideroblastic anemia has been described infrequently in Americans of African descent. The purpose of this study was to characterize iron overload attributable to excessive iron absorption in African Americans.<h4>Patients and methods</h4>In a community hematology and medical oncology practice during the interval 1990 to 1993, we identified and evaluated a series of cases comprised of 6 men and 1 woman, with a mean age of 55 +/- 14 (SD) years (range 33 to 69). Data on clinical features, serum iron parameters, liver and body iron stores, evaluations of anemia, human leukocyte antigen (HLA) typing, and family studies were analyzed.<h4>Results</h4>Among our patients, the serum iron parameters were: iron concentration 26 +/- 13 mumol/L, transferrin saturation 59 +/- 21%, and ferritin concentration 1,588 +/- 1,053 micrograms/L. Clinical abnormalities observed included weakness and fatigue, decreased libido and impotence, hepatopathy, arthropathy, diabetes mellitus, hypogonadotrophic hypogonadism, and hyperpigmentation. Hepatic parenchymal cell iron deposits were increased in each of the 6 patients studied, and Kupffer cell iron deposits were prominent in 4. The occurrence of iron overload was verified by liver iron quantification and therapeutic phlebotomy. Four subjects had alpha-thalassemia minor; 2 others had hemoglobin S and C traits. No proband had HLA-A3 positivity. Four probands had other family members with iron overload.<h4>Conclusions</h4>In comparison with Caucasians with hemochromatosis, our patients have slightly lower mean values of serum iron concentration and transferrin saturation, more Kupffer cell iron deposits, a higher incidence of thalassemia and hemoglobinopathy, and infrequent positivity for HLA-A3. Iron overload in African Americans appears to be more similar to that in certain sub-Saharan African natives than to hemochromatosis.

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 is a putative tumor-suppressor gene. Alternative splicing of transcripts of transmembrane proteins, including N-CAM, is known to occur, resulting in different isoforms of the protein. Using five antibodies against the DCC gene product (three monoclonal antibodies raised in our laboratory, one commercially available antibody, and a rabbit polyclonal antibody), we have demonstrated by immunostaining a DCC protein isoform in reticuloendothelial cells in human thymus, tonsil, and lymph node. This can be distinguished from another isoform described in normal colonic epithelium, because this latter is not demonstrable with the antibodies we have used. It could not be detected in normal colonic epithelium, polyps or colorectal carcinomas. This restrictive distribution suggests that not all DCC gene products are important in colonic cancer.

Astroviruses cause outbreaks of diarrhea in children attending day care centers (DCCs). Reverse transcriptase-polymerase chain reaction (RT-PCR) was compared with EIA detection of astrovirus in stool specimens to characterize further the molecular epidemiology of an outbreak of astrovirus-associated gastroenteritis. Three hundred sixty-eight stool specimens collected prospectively from 36 children enrolled in a DCC during an 11-week outbreak of diarrhea were evaluated by EIA and RT-PCR. Astrovirus was detected in 32% of specimens by RT-PCR versus 10% by EIA (P < .001) and in 89% of children by RT-PCR versus 50% by EIA. The median duration of astrovirus excretion episodes detected by EIA was 1.5 days versus 4 days by RT-PCR (P = .06). Astrovirus was excreted for prolonged periods by immunocompetent children during this outbreak. RT-PCR was more sensitive than EIA for detection of astrovirus in stool specimens and redefined the epidemiology of astrovirus infection in this setting.

Estrogen receptor (ER) and progesterone receptor (PR) contents were determined by biochemical (dextran charcoal-coated (DCC) assay) and immunohistochemical (ICA) methods in biopsies from 145 primary endometrial adenocarcinomas and those with eligible receptor measurements were analyzed with respect to correlations to cancer-specific survival in a multivariate analysis including histopathological characteristics. Median patient follow-up time was 67 months with 18 cancer deaths. The PR-DCC and ER-DCC values were dichotomized according to levels previously found by us to correspond to the best agreement between receptor status as determined by the DCC and ICA methods (130 fmol/mg cytosol protein for ER, 114 fmol/mg for PR). Using these thresholds, we found by multivariate analysis that "high" PR-DCC levels (> 114 fmol/mg) correlated significantly (P = 0.004) with survival, independent of stage risk group (Ia + b vs Ic-IV). Patient age and histologic grade were prognostic factors in a univariate setting, but these parameters were eliminated in the multivariate model. While the PR-ICA scores also correlated significantly and independently with survival, the predictive effect of PR-ICA positivity alone could not be statistically evaluated due to the number of cases with eligible ICA values. However, we suggest that owing to a close correlation between DCC and ICA results, PR-ICA status may provide significant prognostic information when DCC measurements are not available.

Polymorphic (CTC)n and (TAAA)n sequences were identified in exons 1 and 8 of the myelin oligodendrocyte glycoprotein (MOG) gene. The different alleles were detected by a method combining fluorescence labeling of polymerase chain reaction (PCR) products and use of an automated DNA sequencer. Although only two alleles differing by the number of leucine residues encoded by the (CTC)n array were detected at the first locus, seven alleles were identified at the second. The high degree of polymorphism (75%) of the tetranucleotide repeat makes this marker informative for association or linkage studies with diseases such as hemochromatosis or multiple sclerosis.

The Ber-ACT35 mAb was raised against the human T-cell lymphotrophic virus (HTLV) 1-transformed, CD4+ HUT 102 cell line and recognizes the human homologue of the OX40 (hOX40) antigen. The analysis of the expression of hOX40 by immunohistochemical techniques in malignant lymphomas, carcinomas and non-malignant tissues of different organs shows that hOX40 expression was almost completely restricted to T lymphocytes. Besides T cells only a small subpopulation of macrophages in Langerhans' cell histiocytosis and a few blasts in B-cell non-Hodgkin's lymphoma (B-NHL) revealed a faint immunostaining with the Ber-ACT35 mAb. Furthermore, most of the hOX40+ T-cells are CD4+.

Photoreceptor induction in the Drosophila eye is mediated by activation of the Ras signal transduction cascade. Although this process is well understood, little is known about how the diversity of photoreceptor subtypes is generated. The pipsqueak (psq) gene is expressed at high levels in the R3/R4 precursors during eye development and this expression depends on seven-up (svp) gene function. Moreover, strong psq alleles are dominant suppressors of a svp-induced cone cell transformation phenotype. Although the gene was previously identified and described as a member of the maternal posterior group of genes, the strong semilethal alleles isolated here demonstrate a specific requirement for psq function downstream of svp for the development of photoreceptors R3/R4. The gene has three independent 5' ends and codes for several nuclear protein isoforms, some containing the POZ domain which has been implicated in protein-protein interactions. Interestingly, all viable alleles with a maternal posterior group phenotype cluster around one specific 5' exon, while all semilethal alleles have lesions which map to a different alternative 5' exon.

Interference with VP16-mediated activation of herpes virus immediate-early (or alpha) genes is thought to be the major cause of establishing viral latency in sensory neurons. This could be brought about by lack of a key activating transcription factor(s) or active repression. In this study we find that sensory neurons express all important components for VP16-mediated alpha gene induction, such as the POU transcription factor Oct-1, host cell factor (HCF) and GABP alpha/beta. However, Oct-1 and GABP alpha/beta are only present at low levels and the VP16-induced complex (VIC) appears different. We do not find protein expression of the transcription factor Oct-2, implicated by others as an alpha gene repressor. The POU factor N-Oct3 (Brn 2 or POU3F2) is also present in sensory neurons and binds viral TAATGARAT motifs with higher affinity than Oct-1, indicating that it may be a candidate repressor for competitive binding to TAATGARAT motifs. When transfected into HeLa cells, where Oct-1 and GABP alpha/beta are highly abundant, N-Oct3 represses model promoters with multimerized TAATGARAT motifs, but fails to repress complete alpha gene promoters. Taken together our findings suggest that modulation of alpha gene promoters could contribute to viral latency when low concentrations of the activating transcription factors Oct-1 and GABP alpha/beta prevail. Our data, however, refute the notion that competing Oct factors are able to block alpha gene transcription to achieve viral latency.

Loss of DCC gene expression has now been demonstrated in a wide variety of metastatic cancers. Here we present the nucleotide sequence and predicted amino acid sequence of mouse DCC. Mouse and human DCC share 96% identity at the amino acid level. Analysis of DCC mRNA expression throughout the mid and late stages of gestation in the mouse, demonstrated that DCC mRNA is expressed at significantly higher levels in the developing mouse embryo than in any adult tissue. In addition, we show that an embryo-specific, alternatively spliced, form of DCC is expressed in day 9.5 through day 18.5 embryos. The expression of both alternatively spliced forms of DCC is developmentally regulated such that the embryonic form of DCC predominates in day 9.5 and 10.5 embryos. In the later stage embryos the expression of this alternatively spliced form of DCC is down-regulated with respect to that of the adult form. Whole-mount in situ hybridization of day 11.5 mouse embryos revealed that DCC mRNA is expressed at high levels in the developing brain and the neural tube. However, no DCC mRNA could be detected in any other embryonic tissue at this stage of development. These observations suggest that during embryogenesis DCC may play a pivotal role in the development of the central nervous system.

The DCC (deleted in colorectal cancer) candidate tumor suppressor gene spans greater than 1350 kilobases at chromosomes 18q21.1 and encodes a transmembrane protein of unknown function. Although DCC is expressed in a number of adult tissues, its expression is highest in the brain and we have, therefore, undertaken studies to determine if DCC inactivation might contribute to tumors arising there. Decreased or absent DCC protein expression was noted in more than 50% of the thirty brain tumors studied. Although specific mutations in the DCC gene were not identified, a variety of mechanisms appeared to contribute to the altered DCC expression, including allelic loss, aberrant splicing of transcripts and allele-specific loss of transcripts. In total, the data suggest that DCC inactivation may be important in brain tumor pathogenesis.

Haemochromatosis is a common autosomal recessive disorder of iron metabolism caused by a gene in tight linkage with HLA class I genes. Despite intensive research, the molecular defect and underlying biochemical anomaly are still unknown. Diabetes, a serious complication of haemochromatosis, is frequently associated with cirrhosis which reduces life expectancy. Its development is related to iron excess, directly or through associated liver involvement, although the precise mechanisms of iron toxicity remain unclear. New concepts concerning its pathogenesis include insulin resistance and beta-cell dysfunction which are apparent well before insulin deficiency and can be reversed if iron depletion is promptly initiated. Today, earlier recognition of iron overload through active diagnostic approaches has a direct impact in reducing the frequency of diabetes among hemochromatosis patients. Presymptomatic diagnosis in the general population and among relatives of affected subjects currently relies on the detection of increased iron stores through medical awareness and family screening. Indirect gene diagnosis with serological and molecular markers of the HLA region can be provided for relatives of proven cases. As part of a genetic counselling process, this allows the identification of at-risk subjects before the onset of iron accumulation. Isolation of the gene and identification of the metabolic defect leading to increased iron absorption may have significant implications for future diagnostic procedures and preventive strategies in haemochromatosis.

We investigated the evolutionary history of the divergent vertebrate linker histones H1 zero, H5, and H1M. We observed that the sequence of the central conserved domain of these vertebrate proteins shares characteristic features with histone H1 proteins of plants and invertebrate animals which otherwise never appear in any vertebrate histone H1 protein. A quantitative analysis of 58 linker histone sequences also reveals that these proteins are more similar to invertebrate and plant histone H1 than to histone H1 of vertebrates. A phylogenetic tree deduced from an alignment of the central domain of all known linker histones places H1 zero, H5, and H1M in close vicinity to invertebrate sperm histone H1 proteins and to invertebrate histone H1 proteins encoded by polyadenylated mRNAs. We therefore conclude that the ancestors of the vertebrate linker histones H1 zero, H5, and H1M diverged from the main group of histone H1 proteins before the vertebrate type of histone H1 was established in evolution. We discuss this observation in the general context of linker histone evolution.

The effect of dalteparin, a low molecular weight heparin, on severely antithrombin III (ATIII)-decreased disseminated intravascular coagulation (DIC) model was compared with that of unfractionated heparin (heparin). The DIC model in rabbits was produced by continuous infusion of thrombin in combination with bolus injection of latex. After a 3 hr infusion of thrombin, plasma ATIII activity was lowered to 30% of normal plasma. Platelet number, fibrinogen content and alpha 2 plasmin inhibitor (alpha 2PI) activity were also decreased. Dalteparin (25-100 IU/kg/hr) and heparin (25-100 U/kg/hr) inhibited the decrease in ATIII activity, platelet number and fibrinogen content, and had no effect on alpha 2PI activity. Activated partial thromboplastin time (APTT) was prolonged by heparin (50 and 100 U/kg/hr), but not by dalteparin (25-100 IU/kg/hr). The ratio of anti-factor Xa (F.Xa) activity to anti-thrombin activity for dalteparin (50 IU/kg/hr) was higher than that for heparin (50 U/kg/hr). With the addition of exogenous ATIII, the ratio of anti-F.Xa to anti-thrombin for heparin increased, but that for dalteparin did not change. However, the increased ratio for heparin was still lower than the unchanged ratio for dalteparin. These results suggest that both dalteparin and heparin have the ability to rectify the abnormal parameters of severely ATIII-decreased DIC, and that the effects of dalteparin are mainly involved with anti-F.Xa activity whereas the effects of heparin are via anti-thrombin activity.

Regulation of iron balance is of particular interest, especially iron absorption, cellular iron metabolism and transferrin-transferrin receptor in hematopoiesis. Recent advances in molecular and cell biology have helped to reveal the mysteries of cellular iron metabolism concerning mRNA encoding ferritin and transferrin receptor synthesis. The physiology of transferrin and transferrin receptor is applied in the evaluation of erythropoiesis, i.e., erythron transferrin uptake in ferrokinetics and measurement of serum transferrin receptor. In iron absorption, much of the key mechanism remains unknown. The importance of iron metabolism in human beings is discussed in traditional areas of iron deficiency and nutrition. Iron overload is a new clinical problem to be solved in hemochromatosis or in relation to ischemic heart disease.

Treatment of rats in vivo with NGF promotes the survival and enhances the neurotransmitter phenotype of basal forebrain cholinergic neurons. We showed recently (Williams et al., 1993) that NGF-induced stimulations of the cholinergic markers ChAT and high-affinity choline uptake are reflected in an enhanced synthesis and release of ACh in terminals fields of basal forebrain cholinergic neurons. The objective of the present study was to determine whether such effects translate into an enhancement in neurotransmission between nucleus basalis neurons and postsynaptic target cells, and therefore are likely to be of physiological significance. Changes in cholinergic neurotransmission after NGF were assessed by comparing the ability of cholinergic pathway activation, produced by electrical stimulation of nucleus basalis or the external capsule, to elicit intracellularly recorded muscarinic responses in basolateral amygdaloid (BLA) neurons in ventral forebrain slice preparations from NGF-treated and control Fischer 344 adult rats. Chronic infusion of NGF for 3 weeks (1.2 micrograms/d, i.c.v.) increased the likelihood of eliciting cholinergic slow depolarizations (slow EPSP) via stimulation of cholinergic pathways in the slice. In addition, the frequency-response curves for generation of the cholinergic slow EPSP by nucleus basalis or external capsule stimulation were shifted approximately twofold to the left and the EF50 values significantly reduced in neurons from NGF-treated slices, compared to those in preparations from vehicle-treated or untreated controls. Treatment with NGF also resulted in a leftward shift in the frequency-response curve for cholinergic pathway-induced blockade of the slow afterhyperpolarization, without change in the maximal inhibitory effect. The NGF-induced enhancement in cholinergic synaptic effectiveness was not accompanied by alterations in the resting membrane properties or intrinsic excitability of BLA pyramidal neurons. Nor did treatment with NGF affect their chemosensitivity or responsiveness to direct postsynaptic applications of the cholinergic carbachol. We conclude from these results that chronic administration of exogenous NGF can facilitate neurotransmission within basal forebrain cholinergic projections in normal adult brain, presumably as a consequence of its ability to stimulate presynaptic mechanisms involved in synthesis and/or release of ACh.

The inhibition of thrombin generation (TG) was studied in plasma from human volunteers after single subcutaneous administrations of 4000, 8000 or 12,000 anti-Xa units (i.e., 6, 12 or 18 mg) of the synthetic pentasaccharide (SR 90107/ORG 31540) (SP). SP impaired TG in plasma for up to 18 h after injection, and the time-courses of TG and factor Xa inhibitions were similar. In untreated plasma supplemented in vitro with SP to obtain the same anti-Xa activity as in ex vivo samples, equivalent TG inhibitions were observed thus showing that no transformed SP molecules were involved in the TG inhibition ex vivo. Functional as well as immunological assay of TFPI indicated that subcutaneous injection of 12,000 anti-Xa units of SP did not induce any TFPI release, whereas under the same conditions, 13,000 IU of Fraxiparine produced a significant rise of TFPI in plasma. The plotting of TG inhibition versus SP concentration could be fitted with a good correlation (r = 0.94) to the graphical representation linking [ATIII-SP] to [SP]. These results demonstrate that following subcutaneous administration to man, SP inhibits TG ex vivo and likely in vivo exclusively through the same selective ATIII-mediated inhibition of factor Xa as the one elicited in vitro.