Precision measurements of the np scattering differential cross section in the intermediate energy region.

In fast neutron cancer therapy, approximately 50% of the cell damage is caused by recoil protons from neutron-proton (np) scattering. In the intermediate energy region, there is a need for unambiguous np scattering data with good precision in both the shape of the angular distribution and the absolute normalisation. The normalisation techniques have been reviewed for np scattering measurements as well as recent experimental results, particularly the data obtained at The Svedberg Laboratory at 96 and 162 MeV. In addition, to what extent systematic uncertainties in the np differential cross section might affect the determination of proton recoil kerma coefficients is investigated.

Kerma coefficients for neutron scattering on 12C and 16O at 96 MeV.

Recently, many new applications of fast neutrons are emerging or under development, like dose effects due to cosmic ray neutrons for airplane crew, fast neutron cancer therapy, studies of electronics failure induced by cosmic ray neutrons and accelerator-driven incineration of nuclear waste and energy production technologies. In radiation treatment, the kerma (Kinetic energy release in matter) coefficient, which describes the average energy transferred from neutrons to charged particles, is widely used. The kerma coefficient can be calculated from microscopic nuclear data. Nuclear data above 20 MeV are rather scarce, and more complete nuclear data libraries are needed in order to improve the understanding of the processes occurring on a cellular level. About half the dose in human tissue due to fast neutrons comes from proton recoils in neutron-proton (np) scattering, 10-15% from nuclear recoils due to elastic and inelastic neutron scattering and the remaining 35-40% from neutron-induced emission of light ions. Experimental data on elastic and inelastic neutron scattering at 96 MeV from (12)C and (16)O have been obtained recently at The Svedberg Laboratory in Uppsala, Sweden. These data are shown to be relevant for the determination of nuclear recoil kerma coefficients from elastic and inelastic neutron scattering at intermediate energies.

Elastic neutron scattering studies at 96 MeV for transmutation.

Elastic neutron scattering from (12)C, (14)N, (16)O, (28)Si, (40)Ca, (56)Fe, (89)Y and (208)Pb has been studied at 96 MeV in the10-70 degrees interval, using the SCANDAL (SCAttered Nucleon Detection AssembLy) facility. The results for (12)C and (208)Pb have recently been published, while the data on the other nuclei are under analysis. The achieved energy resolution, 3.7 MeV, is about an order of magnitude better than for any previous experiment above 65 MeV incident energy. A novel method for normalisation of the absolute scale of the cross section has been used. The estimated normalisation uncertainty, 3%, is unprecedented for a neutron-induced differential cross section measurement on a nuclear target. Elastic neutron scattering is of utmost importance for a vast number of applications. Besides its fundamental importance as a laboratory for tests of isospin dependence in the nucleon-nucleon, and nucleon-nucleus, interaction, knowledge of the optical potentials derived from elastic scattering come into play in virtually every application where a detailed understanding of nuclear processes is important. Applications for these measurements are dose effects due to fast neutrons, including fast neutron therapy, as well as nuclear waste incineration and single event upsets in electronics. The results at light nuclei of medical relevance ((12)C, (14)N and (16)O) are presented separately. In the present contribution, results on the heavier nuclei are presented, among which several are of relevance to shielding of fast neutrons.

Light charged-particle production in 96 MeV neutron-induced reactions on carbon and oxygen.

In recent years, an increasing number of applications involving fast neutrons have been developed or are under consideration, e.g. radiation treatment of cancer, neutron dosimetry at commercial aircraft altitudes, soft-error effects in computer memories, accelerator-driven transmutation of nuclear waste and energy production and determination of the response of neutron detectors. Data on light-ion production in light nuclei such as carbon, nitrogen and oxygen are particularly important in calculations of dose distributions in human tissue for radiation therapy at neutron beams, and for dosimetry of high-energy neutrons produced by high-energy cosmic radiation interacting with nuclei (nitrogen and oxygen) in the atmosphere. When studying neutron dose effects, it is especially important to consider carbon and oxygen, since they are, by weight, the most abundant elements in human tissue. Preliminary experimental double-differential cross sections of inclusive light-ion (p, d, t, (3)He and alpha) production in carbon induced by 96-MeV neutrons have been presented. Energy spectra were measured at eight laboratory angles: 20, 40, 60, 80, 100, 120, 140 and 160 degrees. Measurements were performed at The Svedberg Laboratory (TSL), Uppsala, using the dedicated MEDLEY experimental setup. The authors have earlier reported experimental double-differential cross sections of inclusive light-ion production in oxygen. In this paper, the deduced kerma coefficients for oxygen has been presented and compared with reaction model calculations.

Spin and fluorescent probing of the binding interface between tissue factor and factor VIIa at multiple sites.

The specific complex between the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa) was chosen as a model for studies of the binding interface between two interacting proteins. Six surface-exposed positions in sTF, residues known to contribute to the sTF-FVIIa interaction, were selected for cysteine mutation and site-directed labeling with spin and fluorescent probes. The binding interface was characterized by spectral data from electron paramagnetic resonance (EPR) and steady-state and time-domain fluorescence spectroscopy. The labels reported on compact local environments at positions 158 and 207 in the interface region between sTF and the gamma-carboxyglutamic acid (Gla) domain of FVIIa, and at positions 22 and 140 in the interface region between sTF and the first epidermal growth factor-like (EGF1) domain of FVIIa. The tightness of the local interactions in these parts of the interface is similar to that seen in the interior of globular proteins. This was further emphasized by the reduced local polarity detected by the fluorescent label upon FVIIa binding, especially in the sTF-Gla region. There were indications of structural rigidity also at positions 45 and 94 in the interface region between sTF and the protease domain (PD) of FVIIa, despite the perturbed cofactor function of these sTF variants. The results of the present study indicate that the multi-probing approach enables comparison of the tightness and characteristics of interaction along the binding interface of a protein complex. This approach also increases the probability of acquiring reliable structural data that are descriptive of the wild-type proteins.

Estrogen receptors in the human forebrain and the relation to neuropsychiatric disorders.

The steroid hormone estrogen influences brain function and neuropsychiatric disorders, but neuroanatomical information about the estrogen receptors (ERs) are rather limited. The main focus of this article is to provide an overview of the current status of the ER distribution and possible function in the human brain. The ERs are ligand activated transcription factors that belong to the steroid hormone receptors, included in the nuclear receptor superfamily. To date, there are two known ER subtypes, alpha and beta. In the human forebrain, both estrogen receptor subtypes are predominantly expressed in limbic-related areas, although they show distinct distribution patterns. The ERalpha mRNA expression appears to dominate in the hypothalamus and amygdala, indicating that the alpha-subtype might modulate neuronal cell populations involved in autonomic and reproductive neuroendocrine functions as well as emotional interpretation and processing. In contrast, the hippocampal formation, entorhinal cortex, and thalamus appear to be ERbeta dominant areas, suggesting a putative role for ERbeta in cognition, non-emotional memory and motor functions. Clinical observations of estrogenic effects together with the information available today regarding ER expression in the primate brain provide important clues as to the functional aspects of the two ER subtypes. However, further characterization of the different phenotypes of the ER expressing cells in the human brain is needed as well as the delineation of the genes which are regulated by the ERs and how this transcriptional control correlates with human behavior and mental status.

Estrogen receptor beta (ERbeta) messenger ribonucleic acid (mRNA) expression within the human forebrain: distinct distribution pattern to ERalpha mRNA.

Estrogen has been shown to influence several brain functions as well as the expression of neuropsychiatric diseases. To date, two estrogen receptor (ER) subtypes have been identified, ERalpha and ERbeta. ERalpha messenger ribonucleic acid (mRNA) distribution in the human forebrain was recently characterized, and the highest expression was found in restricted areas of the amygdala and hypothalamus. However, no information exists with regard to ERbeta mRNA distribution in the human brain. To this end, the anatomical distribution pattern of ERbeta mRNA expression in the human forebrain was investigated in the present study. Overall, the ERbeta mRNA hybridization signal was relatively low, but the most abundant ERbeta mRNA areas were the hippocampal formation (primarily the subiculum), claustrum, and cerebral cortex; expression was also present in the subthalamic nucleus and thalamus (ventral lateral nucleus). In contrast to ERalpha (studied on adjacent brain sections), ERbeta mRNA expression was low in the hypothalamus and amygdala. Based on the revealed anatomical distribution of the human ERbeta gene expression, a putative role for ERbeta in the modulation of cognition, memory, and motor functions is suggested.

The human brain has distinct regional expression patterns of estrogen receptor alpha mRNA isoforms derived from alternative promoters.

The human estrogen receptor (ER) alpha gene is transcribed from multiple promoters, generating mRNA isoforms with unique 5' ends in the untranslated region. In the present study, alternative promoters were shown to regulate the ERalpha gene expression in different neuronal populations of the human brain. By using in situ hybridization histochemistry, the A and B promoters, but not the C promoter, in the ERalpha gene were found to be active in the human forebrain. The mRNA isoform transcribed from the A promoter was expressed in low levels in most of the brain areas where ERalpha mRNA was present. In contrast, the B promoter mRNA isoform was more restricted, localized predominantly in high-expressing ERalpha mRNA regions. The gross anatomical distribution of the different mRNA isoforms analyzed with RT-PCR generally supported the results obtained by the in situ hybridization. Estrogen is known to modulate many different brain functions, such as neuroendocrine events associated with reproduction, mood, and cognition, likely to be mediated by different neuronal populations. Thus, the current findings of alternative ERalpha promoter expression in distinct neuronal populations suggest that multiple promoter usage is a possible mechanism to achieve differentiated regulation of the ERalpha expression, dependent on the cell phenotype and consequently the functions mediated by the specific neuron.

Targeted destabilization of HY5 during light-regulated development of Arabidopsis.

Arabidopsis seedlings display contrasting developmental patterns depending on the ambient light. Seedlings grown in the light develop photomorphogenically, characterized by short hypocotyls and expanded green cotyledons. In contrast, seedlings grown in darkness become etiolated, with elongated hypocotyls and dosed cotyledons on an apical hook. Light signals, perceived by multiple photoreceptors and transduced to downstream regulators, dictate the extent of photomorphogenic development in a quantitative manner. Two key downstream components, COP1 and HY5, act antagonistically in regulating seedling development. HY5 is a bZIP transcription factor that binds directly to the promoters of light-inducible genes, promoting their expression and photomorphogenic development. COP1 is a RING-finger protein with WD-40 repeats whose nuclear abundance is negatively regulated by light. COP1 interacts directly with HY5 in the nucleus to regulate its activity negatively. Here we show that the abundance of HY5 is directly correlated with the extent of photomorphogenic development, and that the COP1-HY5 interaction may specifically target HY5 for proteasome-mediated degradation in the nucleus.

The human forebrain has discrete estrogen receptor alpha messenger RNA expression: high levels in the amygdaloid complex.

Estrogen is considered to play an important role in neuropsychiatric disorders and the estrogen receptors mediate the action of the hormone. In the present study, the messenger RNA expression pattern of the estrogen receptor alpha subtype was identified in the post mortem human brain. High stringent in situ hybridization histochemistry was performed using a riboprobe specific for the estrogen receptor alpha subtype. The human brain was mainly characterized by abundant estrogen receptor alpha messenger RNA expression in the amygdala and hypothalamus, but labeling (lower) was also found in the extended sublenticular amygdala, cerebral cortex, and hippocampus. In the amygdala, the estrogen receptor alpha messenger RNA was preferentially expressed in medially-localized nuclei suggesting that estrogen regulates distinct human amygdala-mediated functions. The Cynomologous monkey brain was also examined in the present study and a similar distribution of the estrogen receptor alpha messenger RNA signal was observed in the human and monkey brain. However, the primate expression pattern differed in part from the known distribution in the rat. The current results show that estrogen receptor alpha messenger RNA is expressed in discrete areas of the human brain not only related to neuroendocrine function, but also emotion, memory, and cognition, which is consistent with the hypothesized involvement of estrogen in schizophrenia, affective disorders, and Alzheimers disease.

Effects of chronic 17beta-estradiol treatment on the serotonin 5-HT(1A) receptor mRNA and binding levels in the rat brain.

Acute 17beta-estradiol treatment had been shown to downregulate the 5-HT(1A) receptor mRNA expression in limbic areas of the female rat brain. The aim of the present study was to determine the effects of chronic 17beta-estradiol treatment on 5-HT(1A) receptor mRNA expression and 5-HT(1A) receptor binding in ovariectomized female rats. Using in situ hybridization histochemistry, no alterations were found on the 5-HT(1A) receptor mRNA levels after the estradiol treatment (2 weeks). Radioligand autoradiographic studies using the selective 5-HT(1A) receptor antagonist [(3)H]WAY-100635 revealed reduced receptor binding in the amygdala, hippocampus, perirhinal cortex, and motor cortex after estradiol treatment, whereas no changes were observed in the piriform or retrosplenial cortex. Thus, the previous findings together with the present results indicate that estradiol-induced alterations in 5-HT(1A) receptor mRNA expression appears within hours, but diminishes with chronic treatment when significant changes on the receptor-protein level are apparent. The effects of estradiol treatment on the 5-HT(1A) receptor binding in the limbic areas suggest that estrogen can modulate functions such as learning, memory, cognition, emotional processing, and social behavior. Consequently, estradiol modulation of 5-HT(1A) receptor circuits might be a possible pathway for the estrogen influence in the expression of psychiatric and neurological disorders such as Alzheimer's disease, affective disorders, and schizophrenia.

Properties of spin and fluorescent labels at a receptor-ligand interface.

Site-directed labeling was used to obtain local information on the binding interface in a receptor-ligand complex. As a model we have chosen the specific association of the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), the primary initiator of the blood coagulation cascade. Different spectroscopic labels were covalently attached to an engineered cysteine in position 140 in sTF, a position normally occupied by a Phe residue previously characterized as an important contributor to the sTF:FVIIa interaction. Two spin labels, IPSL [N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide] and MTSSL [(1-oxyl-2,2,5, 5-tetramethylpyrroline-3-methyl)methanethiosulfonate], and two fluorescent labels, IAEDANS [5-((((2-iodoacetyl)amino) ethyl)amino)naphthalene-1-sulfonic acid] and BADAN [6-bromoacetyl-2-dimethylaminonaphthalene], were used. Spectral data from electron paramagnetic resonance (EPR) and fluorescence spectroscopy showed a substantial change in the local environment of all labels when the sTF:FVIIa complex was formed. However, the interaction was probed differently by each label and these differences in spectral appearance could be attributed to differences in label properties such as size, polarity, and/or flexibility. Accordingly, molecular modeling data suggest that the most favorable orientations are unique for each label. Furthermore, line-shape simulations of EPR spectra and calculations based on fluorescence depolarization measurements provided additional details of the local environment of the labels, thereby confirming a tight protein-protein interaction between FVIIa and sTF when the complex is formed. The tightness of this local interaction is similar to that seen in the interior of globular proteins.

The flinders sensitive line rats, a genetic model of depression, show abnormal serotonin receptor mRNA expression in the brain that is reversed by 17beta-estradiol.

The possible link between estrogen and serotonin (5-HT) in depression was investigated using a genetic animal model of depression, the Flinders Sensitive Line (FSL) rats, in comparison to control Flinders Resistant Line rats. The mRNA levels of the estrogen receptor (ER) alpha and beta subtypes and the 5-HT(1A) and 5-HT(2A) receptors were analyzed in several limbic-related areas of ovariectomized FSL and FRL rats treated with 17beta-estradiol (0.15 microg/g) or vehicle. The FSL animals were shown to express significantly lower levels of the 5-HT(2A) receptor transcripts in the perirhinal cortex, piriform cortex, and medial anterodorsal amygdala and higher levels in the CA 2-3 region of the hippocampus. The only significant difference between the rat lines in ER mRNA expression was found in the medial posterodorsal amygdala, where the FSL rats showed lower ERalpha expression levels. Overall, estradiol treatment increased 5-HT(2A) and decreased 5-HT(1A) receptor mRNA levels in several of the examined regions of both lines. Thus, in many areas, estradiol was found to regulate the 5-HT receptor mRNA expression in the opposite direction to the alterations found in the FSL rats. These findings further support the implication of 5-HT receptors, in particular the 5-HT(2A) subtype, in the etiology of affective disorders. Moreover, the ability of estradiol to regulate the expression of the 5-HT(1A) and 5-HT(2A) receptor genes might account for the reported influence of gonadal hormones in mood and depression.

Acute 17 beta-estradiol treatment down-regulates serotonin 5HT1A receptor mRNA expression in the limbic system of female rats.

In situ hybridization histochemistry was used to investigate acute estrogen effects on serotonin 5HT1A receptor mRNA levels in limbic-related brain areas in the female ovariectomized rat. Acute administration of 17 beta-estradiol (10 micrograms) decreased 5HT1A receptor mRNA expression levels within the medial amygdala (after 2 and 24 h), piriform cortex (after 2 and 24 h), and perirhinal cortex (after 24 h). No changes in 5HT1A mRNA levels were observed in hippocampus or retrosplenial cortex. The findings suggest specific regional effects of estrogen on 5HT functions mediated through regulation of the 5HT1A gene.

Differential distribution and regulation of estrogen receptor-alpha and -beta mRNA within the female rat brain.

In the present study, estrogen receptor (ER)alpha and ER beta genes were found to be differentially expressed in discrete subregions of the rat amygdaloid complex. The amygdala nuclei showing predominant ER alpha mRNA expression included the posterolateral cortical nucleus, amygdala hippocampal area, and lateral dorsolateral nucleus, whereas the amygdala areas with predominant ER beta mRNA expression were the medial anterodorsal and central nuclei. Both ER alpha and ER beta mRNAs were highly expressed in the medial posterodorsal nucleus. In addition to the discrete anatomical expression patterns, there appeared to be a differential regulation by estradiol of the ER alpha and ER beta mRNAs. Two weeks of estradiol (170 microgram total) treatment decreased ER alpha mRNA expression levels in the arcuate, ventromedial hypothalamus, and posterolateral cortical amygdala nucleus, but increased ER beta mRNA in the arcuate. In the medial amygdala nuclei, only ER beta mRNA levels were altered (reduced) by estradiol treatment. These results suggest that estrogen can modulate behaviors and functions mediated by the amygdala and hypothalamus via differentially regulated ER subtypes.

Serum leptin concentrations in a normal population and in GH deficiency: negative correlation with testosterone in men and effects of GH treatment.

OBJECTIVE: To study relationships between leptin and factors regulating body composition as well as metabolic risk factors. Furthermore, to study the effects of GH on leptin. DESIGN: Cross-sectional and population-based. Regarding the effects of GH, prospective and interventional. PATIENTS: One hundred and eleven women and 107 men, 20-70 years old, randomly selected from the population registry in the community of Linköping, Sweden. Ten GH-deficient subjects were given GH until normalization of IGF-I levels. MEASUREMENTS: Venous blood was drawn in the fasting state. Serum leptin and hormones were analysed by immunoassay. RESULTS: In the population sample the natural logarithm of leptin (in(leptin)) correlated with body mass index (BMI) (men, r = 0.67), P < 0.0001; women, r = 0.71, P < 0.0001). The median value of leptin was 4.6 micrograms/l in men and 12.3 micrograms/l in women (P < 0.0001). Levels of in(leptin) did not correlate with plasma neuropeptide Y (men, P = 0.13; women, P = 0.35). In men only there was an inverse relationship between in(leptin) and testosterone (r = -0.46, P < 0.0001, after correction for BMI standardized r = -0.26, P = 0.03) as well as IGF-I (r = -0.20, P = 0.048). Although BMI was similar, smoking men had higher leptin levels than non-smoking men (median, 6.6 and 4.2 micrograms/l, respectively; Mann-Whitney; P = 0.006). In the GH-deficient subjects leptin levels were elevated and, although GH treatment did not change BMI, leptin levels decreased (median before GH, 21 micrograms/l and after 15 micrograms/l, respectively; P = 0.017). CONCLUSION: Serum leptin concentration is closely associated with BMI in the population with a gender difference in absolute levels and a strong negative correlation with testosterone in men. Serum leptin is elevated in GH deficiency and lowered by GH substitution.

Analysis of polyomavirus enhancer-effect on DNA replication and early gene expression.

The polyomavirus enhancer is located adjacent to the origin of DNA replication and the transcriptional promoters. It has a cis-acting essential function in the initiation of both viral DNA synthesis and early transcription. The enhancer is activated by the binding of protein factors to specific sites in DNA. Mutants with deletions of the A- or the B-segment of the enhancer were constructed. In mouse 3T6 cells, the transcription of the viral early region was significantly decreased by deletion of the A-segment, but not by deletion of the B-segment. In contrast, the two deletions had a similar, moderately negative effect on viral DNA synthesis. However, the presence of DNA with a wild-type enhancer in doubly transfected cells resulted in very strong interference with the replication, but not with the transcription, of deletion mutant DNA. DNA of the deletion mutants were subjected to site-directed mutagenesis of the remaining enhancer segment. Three non-viable mutants were isolated. All three had base-pair changes in the A-segment affecting immediately adjacent binding sites of cellular protein factors. The mutants had lost the enhancer activity on the early promoter, but only one of them with multiple base substitutions had lost the capacity of DNA replication. Together, the results suggest that different aspects of enhancer function determine the activity in initiation of transcription and replication.

Mutational analysis of DNA sequences affecting the replication of defective polyomavirus variant D-50.

The genome of the defective polyomavirus variant D-50 consists of tandemly repeated DNA segments. The repeat unit corresponds to a 17% fragment of polyomavirus DNA (Griffin and Fried (1975) Nature 256, pp. 175-179). To allow mutational analysis, a monomer unit of D-50 DNA was cloned. After excision from the plasmid and ligation to form a random mixture of products, circular head to tail oligomers of the cloned segment were replicated in transfected cells. Those molecules had the same replication properties as original D-50 DNA. Deletion of base sequences assumed to be at the origin of DNA synthesis inhibited the replication completely, whereas a deletion of a segment at the junction of the tandem repeats had only a slight inhibitory effect. Mutation of potential coding sequences of the variant genome had a slight stimulatory effect on DNA synthesis, ruling out that D-50 expresses any protein that stimulates replication. In an attempt to construct variants similar to D-50, cells were transfected with polyomavirus DNA fragments including the sequences of the D-50 monomer unit. However, all these molecules replicated very slowly, suggesting the presence of an element that inhibited DNA synthesis. The combined data show that D-50 genomes can be reconstituted by ligation of monomer units and that the origin of DNA replication was the only essential element of the variant genome, whereas other elements had cis-acting auxiliary functions.

Inhibition of polyoma DNA synthesis by base pair substitutions at the replication origin.

The effect of base pair substitutions on the function of the polyoma virus origin of DNA replication was studied. The mutations were all C-G to T-A transitions, induced by bisulfite treatment of recombinant DNA molecules. The mutagenesis was directed to short single-stranded gaps in duplex DNA, or to loops in heteroduplex molecules. Modification of a 34 base pair sequence of dyad symmetry led to cis-acting inhibition of viral DNA synthesis, ranging from slight defects to total inactivation. One of the mutants was temperature sensitive. Mutants with base changes in an adjacent DNA segment, including an 18 base pair long purine-pyrimidine tract, had similar, but less severe, deficiences. In contrast to the effect of mutations in the homologous region of the simian virus 40 genome, there was no strict relationship between mutation of the putative large T-antigen-binding base sequence GPuGGC and defective viral DNA synthesis.