Transfusion-transmitted infectious diseases.

A spectrum of blood-borne infectious agents is transmitted through transfusion of infected blood donated by apparently healthy and asymptomatic blood donors. The diversity of infectious agents includes hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency viruses (HIV-1/2), human T-cell lymphotropic viruses (HTLV-I/II), Cytomegalovirus (CMV), Parvovirus B19, West Nile Virus (WNV), Dengue virus, trypanosomiasis, malaria, and variant CJD. Several strategies are implemented to reduce the risk of transmitting these infectious agents by donor exclusion for clinical history of risk factors, screening for the serological markers of infections, and nucleic acid testing (NAT) by viral gene amplification for direct and sensitive detection of the known infectious agents. Consequently, transfusions are safer now than ever before and we have learnt how to mitigate risks of emerging infectious diseases such as West Nile, Chikungunya, and Dengue viruses.

The brain-heart connection in mitochondrial respiratory chain diseases.

Mitochondrial respiratory chain disorders (MRCD) are a heterogeneous group of diseases leading to an inadequate production of ATP. Brain and heart are among the most affected organs. Thus far, no specific relationship has been found between specific affected areas in the central nervous system and cardiac involvement. This study investigated the relationship between specific brain involvement and heart disease in mitochondrial disorders. We hypothesize that specific areas of brain lesions in children with MRCD are more frequently correlated to heart disease than others. A retrospective evaluation of the clinical records of 63 children with a definite MRCD, was performed searching for heart disease, namely, dilated and hypertrophic cardiomyopathy and arrhythmia. Brain MR images were evaluated and characterized regarding specific areas of atrophy and involvement. These findings were correlated using the Fischer exact test whose strength was determined with the Phi coefficient. During the period analyzed, 13 children (20.6%) developed cardiac disease, of whom nine (14.3%) exhibited isolated cardiomyopathy, one (1.6%) exhibited arrhythmia and three both. The main MRI abnormalities observed were brain atrophy (65.1%) and among this group 17.5% of subjects had cerebellar involvement. In addition, supratentorial, cerebellar and brainstem white and grey matter lesions were also found. There was a statistically significant relationship between progression to cardiac disease and cerebellar atrophy (Fisher's Exact Test -0.005 and Phi 0.394) and lesions in the cerebral peduncles (0.035/0.358). Moreover, there was an additional correlation between thalamic lesions and progression to hypertrophic myocardiopathy (0.029/0.397). A statistical relationship between thalamic, mesencephalic and cerebellar involvement and cardiac disease in children with definite MRCD was observed. The true significance of this connection warrants further assessment.

Expression of the orphan receptor TR4 during brain development of the rat.

The orphan receptor TR4, member of the nuclear hormone receptor family, is related to the orphan receptors TR2, COUP-TFI and ARP-1, and was originally cloned from the adult rat brain. The latter two orphan receptors have been implicated in central nervous system (CNS) development. To investigate a possible role for TR4 in brain development, expression of TR4 was studied in rat embryos. At embryonic days 14.5 and 19.5, high expression of TR4 was found in the CNS, while low expression was detected throughout the embryo. In postnatal rats, TR4 was mainly expressed in the hippocampus and cerebellum, resembling the expression pattern found in adult brain. These data show that like COUP-TFI and ARP-1, expression of TR4 becomes restricted to distinct areas. In adult brain, TR4 is predominantly expressed in granule cells of both hippocampus and cerebellum. The data suggest a possible role for TR4 during proliferation and maturation of brain structures.

Hypothalamic transcription factors and the regulation of the hypothalamo-neurohypophysial system.

The transcription factors that confer high level expression and regulate the genes encoding neurohypophysial hormones are largely unknown. A number of different approaches have been taken to identify these factors and to elucidate molecular mechanisms of physiological gene regulation. In this chapter two transcription factor families are considered: homeodomain proteins and nuclear receptors. Their identification in the hypothalamus and actions on the OT gene are addressed here.

The nuclear hormone-receptor family in the brain: classics and orphans.

Nuclear hormone receptors comprise a superfamily of over 40 transcription factors. About half of them are classical receptors for lipophilic ligands such as steroids and vitamins. Almost all of these true receptors are present in the brain, where they transduce chemical signals from endocrine organs or signals of nutritional origin into cellular responses. The other members resemble the classical receptors in structure, but have no known ligands, and are hence called 'orphan receptors'. The issue of whether ligands for nuclear orphan receptors exist is controversial. Evidence is emerging that orphan receptors might be activated by signal transduction pathways or might be constitutive enhancers or repressors that interact with the classical receptors. Thus, nuclear orphan receptors are placed in strategic positions in the regulation of gene expression in the nervous system.

Expression of nuclear hormone receptors in the rat supraoptic nucleus.

To investigate the role of nuclear hormone receptors on neuropeptide gene expression in the hypothalamo-neurohypophyseal system (HNS) of the rat, a survey was made of members of the nuclear hormone receptor superfamily that are expressed in the supraoptic nucleus (SON). A polymerase chain reaction cloning strategy based on homologies in the DNA-binding domain of AGGTCA-binding factors was devised for the identification of receptors in microdissected SON tissue. Cloning of the amplified products led to the identification of five true receptors, thyroid hormone receptor-alpha (THR alpha), retinoic acid receptor-alpha, retinoic acid receptor-gamma, retinoid X receptor-alpha, and retinoid X receptor-gamma, as well as four orphan receptors, apolipoprotein AI regulatory protein (ARP-1), chicken ovalbumin upstream promoter transcription factor I (COUP-TF I), estrogen-related receptor 2, and testis receptor 4 (TR4). Dot-blot screening of amplified gene fragment analysis showed that THR alpha, ARP-1, TR4, and COUP-TF I were the most abundant factors expressed in the SON region, in the order THR alpha > ARP-1 > TR4 approximately COUP-TF I. THR alpha has previously been localized to HNS neurons. In situ hybridization analysis showed that ARP-1, COUP-TF I, and TR4 were not expressed in magnocellular neurons at appreciable levels, but rather in surrounding structures. Furthermore, in lactating female rats there were no significant differences in the composition of the nine identified nuclear hormone receptors in the SON region compared with control animals. From these experiments, it is concluded that there is a multitude of hypothalamically expressed nuclear hormone receptors, but that only THR alpha is expressed at relatively high abundance in HNS neurons. This indicates that the peptide-producing magnocellular neurons of the SON express a specific set of transcription factors of the nuclear hormone receptor family.

Localization of transcripts of the related nuclear orphan receptors COUP-TF I and ARP-1 in the adult mouse brain.

The chicken ovalbumin upstream promoter transcription factor, COUP-TF I, and the protein ARP-1 (COUP-TF II) are two highly homologous orphan receptors of the nuclear hormone receptor family. In this study we investigated their expression patterns in the adult nervous system of the mouse. In situ hybridizations were performed on brain sections with 35S-labeled cRNA probes derived from the 3'-non-coding regions of the mARP-1 and mCOUP-TF I mRNAs. Both COUP-TF I and ARP-1 were shown to be expressed in the adult brain and they displayed restricted and distinct expression patterns. COUP-TF I transcripts were predominantly found in the rostral and caudal parts of the adult mouse brain, whereas ARP-1 transcripts prevaled in the middle part of the brain. High expression of COUP-TF I was detected in the olfactory nucleus, in neocortex layers I/II and V/VL, in the dentate gyrus and in areas CA1/CA3/CA4 of the hippocampus, and in the granular layer of the cerebellum. Only low amounts of COUP-TF I mRNA were detected in the ventral, the laterodorsal and in the interanteromedial thalamic nuclei. Small amounts of COUP-TF I transcripts were also found in the epithelial layer of the ventricle and in arachnoid membranes. High expression of ARP-I was detected in the reticular, the ventral lateral and the gelatinosus thalamic nuclei. Other hot spots of ARP-1 mRNA expression were the amygdaloid nucleus and the arachnoid membranes. Lower amounts of ARP-1 transcripts were found in the anterior and lateral hypothalamic areas, in the suprachiasmatic nucleus, and in the choroid plexus.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional domains in the oxytocin gene for regulation of expression and biosynthesis of gene products.

In the oxytocin (OT) gene several regions can be discerned that have a function in regulating its expression. Firstly, in the proximal 5' flanking region regulatory elements have been discovered that are targets for transcription factors of the nuclear hormone receptor family. Through these elements the OT gene of rat and man is responsive to estrogens, thyroid hormones and retinoids. Furthermore, these elements can be employed by the nuclear hormone orphan receptor family for repressive or inductive actions. In the distal 5' flanking region the POU class III proteins Brn-1, Brn-2, Brn-4, that are expressed in magnocellular neurons, and Oct-6 are able to bind, but do not display a significant regulatory activity on the OT gene in heterologous expression systems. Secondly, the OT precursor harbours both the biologically active hormone and the protein neurophysin that is able to associate with the hormone. Heterologous expression of wild-type and mutant vasopressin cDNAs in peptidergic cell lines shows that the highly homologous vasopressin-associated neurophysin domain associates with the hormone domain within the prohormone. This intramolecular interaction between two prohormone domains serves an essential intracellular function, i.e. the proper sorting of the prohormone into the regulated secretory pathway.

Functional analysis of an alternatively spliced estrogen receptor lacking exon 4 isolated from MCF-7 breast cancer cells and meningioma tissue.

An alternatively spliced mRNA coding for a variant estrogen receptor (ER) missing exon 4 (ERdelta4) was detected in the breast tumor cell line MCF7 and meningioma tissue by using the reversed transcriptase PCR technique. The trans-activational properties of this mutant ER were assessed in embryo carcinoma P19EC and human choriocarcinoma JEG3 cells by co-transfection of the ERdelta4 expression vector with an oxytocin promoter construct containing an estrogen-responsive element. ERdelta4 did not trans-activate the oxytocin promoter in either a hormone-dependent or -independent manner. Co-transfection of ERdelta4 together with the wtER did not show any interference of ERdelta4 on the stimulation of the oxytocin promoter by the wtER. ERdelta4 was translated in vitro. Its capacity to bind estradiol, and the binding of the variant to a synthetic estrogen-responsive element were compared to those of the wild-type receptor. ERdelta4 did not bind to a synthetic estrogen-responsive element, nor did it bind estradiol. Hence, ERdelta4 appears to be a silent variant and we speculate that it is without any role in tumor progression.

Repression of estrogen-dependent stimulation of the oxytocin gene by chicken ovalbumin upstream promoter transcription factor I.

The orphan receptor chicken ovalbumin upstream promoter transcription factor I (COUP-TF I) fully prevented not only the activation of the oxytocin gene by retinoic acid and thyroid hormone but also completely repressed the estrogen-dependent stimulation in transfected P19 EC cells. DNase I footprinting showed that the COUP-TF I protein bound to the 5'-flanking region of the oxytocin gene at the site of the distal composite hormone response element, which mediates the responses to estrogen, retinoic acid, and thyroid hormone. Electrophoretic mobility shift assay using this composite hormone response element as probe showed that COUP-TF I and the estrogen receptor competed for binding but did not form a heterodimer. The binding by COUP-TF I was stronger than the binding of the estrogen receptor. Thus, the mechanism of repression involves occupancy of integrated binding sites. By mutagenesis of the composite hormone response element, the COUP-TF I binding site and the estrogen response element could be separated, resulting in functional dissociation of the repressive action of COUP-TF I and the induction by estrogen. The results show that repression of gene expression by COUP-TF I is not limited to receptors that act through heterodimerization but also extends to the homodimer-forming estrogen receptor in a context-dependent manner. This interaction between COUP-TF I and the estrogen receptor may provide a physiological mechanism of selective antagonism of gene regulation by estrogens.

The human vasopressin-oxytocin gene family: no evidence for additional neurophysin-related genes.

Over the last 20 years several observations at the peptide level have indicated the possible existence of additional members of the vasopressin (VP)-oxytocin (OT) gene family in mammals. In this study, the human genome was analyzed for the existence of genes structurally related to the VP and OT genes. Human genomic blots probed under low stringency conditions with exon B of the human OT gene, that codes for the conserved constant region of neurophysin, revealed the presence of two distinct bands in addition to the known VP and OT gene fragments. Five clones were obtained from a library of genomic EcoRI fragments ranging from 4-8 kb, that comprised both low stringency signals, by low stringency hybridization with the OT exon B probe. One clone of 7 kb hybridized at high stringency conditions to bands of the same size as previously detected with OT exon B on a human genomic blot. However, no similarity was observed between the open reading frames of this clone and the neurophysin portion of the OT gene. Another clone of 4.8 kb was identical to a fragment of the gene for the human bone morphogenetic factor hBMP-6, a member of the TGF-beta family. The hBMP-6 gene was not detected by low stringency hybridization of the human genomic blot with the OT exon B probe. No significant similarity was found between the amino acid sequences of human OT neurophysin and hBMP-6. Therefore, no evidence can be provided that the human genome contains additional neurophysin-related genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Wnt-3, a gene activated by proviral insertion in mouse mammary tumors, is homologous to int-1/Wnt-1 and is normally expressed in mouse embryos and adult brain.

We have isolated a common insertion site, Wnt-3, for proviruses of the mouse mammary tumor virus (MMTV). Of mammary tumors induced by the GR variant of MMTV, 5% contains a provirus at Wnt-3, which is located on mouse chromosome 11. The gene is transcribed into a 3.8-kilobase (kb) mRNA in tumors with nearby proviral insertions but not in tumors with proviruses at other loci or in most adult tissues. Normal expression of Wnt-3 is detected in mouse embryos (with a peak around day 12 of gestation) and at low levels in adult brain. The transcriptional unit of the Wnt-3 gene spans approximately 55 kb, with a first intron of 36 kb. The deduced amino acid sequence of the Wnt-3 protein is 47% identical to the int-1/Wnt-1 gene product.

Expression of growth-associated protein B-50 (GAP43) in dorsal root ganglia and sciatic nerve during regenerative sprouting.

Recently it has been shown that B-50 is identical to the neuron-specific, growth-associated protein GAP43. The present study reports on the fate of B-50/GAP43 mRNA and B-50/GAP43 protein, determined by radioimmunoassay, in a rat model of peripheral nerve regeneration (sciatic nerve crush) over a period of 37 and 312 d, respectively. Moreover, the effects of repeated subcutaneous injection of the neurotrophic peptide Org.2766 (an ACTH4-9 analog) and of a conditioning lesion on B-50/GAP43 protein levels in the regenerating nerve and dorsal root ganglia (DRG) were investigated. Both treatments enhanced the functional recovery as evidenced by a foot-flick withdrawal test. Immunocytochemical analysis using antineurofilament antibodies revealed a peptide-induced increase in the number of outgrowing sprouts in the sciatic nerve. Both the peptide and the conditioning lesion amplified the crush lesion-induced increase in B-50 protein content in the nerve as determined by radioimmunoassay. B-50 protein levels seem to correlate proportionally with the number of sprouts. In the DRG of the crushed sciatic nerve, the time course of B-50 expression was studied. B-50 mRNA was quantified from Northern blots. A linear increase up to 10 times the basal level of B-50 mRNA was observed 2 d postsurgery, followed by a gradual decline to normal levels at day 37. The first significant rise in B-50 mRNA level became apparent between 8 and 16 hr after placement of the crush lesion. The first significant rise in B-50 protein level occurred 40 hr after the crush lesion, reaching a plateau of 3 times the basal level between day 6 and 20. B-50 protein levels in DRG cell bodies remained elevated up to 60 d after crush, a period much longer than that observed for B-50 mRNA. Thus, during a later phase of peripheral axonal regeneration, the presence of B-50 appears to be prolonged, probably by an increase in half-life and not so much by enhanced transcription. Treatment with Org.2766 did not affect the B-50/GAP43 levels in DRG cell bodies during the first 6 d following crush. Conditioning lesion resulted in a DRG B-50/GAP43 protein amount at the same level as in rats 14 d after the test lesion. B-50/GAP43 levels in DRG are probably influenced by the rapid axonal transport of the protein, as has been reported by others.