Model-based Analysis for Real-time Label-free Ultraviolet Quantification of Ultrafast Plasmonic Polymerase Chain Reaction.

A mathematical model for DNA quantification was calibrated using experimental results from real-time 260nm absorption measurements of plasmonic PCR thermocycling. The effect of different PCR parameters on template amplification was investigated using the calibrated model.

Molecular studies of a patient with complete androgen insensitivity and a 47,XXY karyotype.

A phenotypic female with complete androgen insensitivity from a maternally inherited mutation in the androgen receptor had a 47,XXY karyotype. Partial maternal X isodisomy explained the expression of androgen insensitivity despite the presence of 2 X chromosomes.

Androgen receptor CAG repeat length contraction in diseased and non-diseased prostatic tissues.

To investigate contraction of CAG repeats within the androgen receptor gene (AR) as shorter CAG repeats have been implicated as a possible risk factor in prostate cancer (PCa). AR CAG repeat lengths were analyzed in DNA from microdissected diseased prostates, leukocytes from matched peripheral blood, and control non-diseased prostates. Consistently, all prostatic tissues, whether from benign or cancerous areas of diseased prostates, or from control prostates, showed multiple AR CAG repeat contractions. Germline DNA from blood leukocytes had single CAG repeat lengths in the normal range. AR CAG repeat length contraction may be involved in prostate carcinogenesis and may precede the pathological process.

Progress in Spinobulbar muscular atrophy research: insights into neuronal dysfunction caused by the polyglutamine-expanded androgen receptor.

Spinobulbar muscular atrophy (SBMA, Kennedy's disease) results from the dysfunction and degeneration of specific motor and sensory neurons. The underlying cause of this ligand-dependent neurodegenerative disease is expansion of the CAG trinucleotide repeat in the androgen receptor (AR) gene which leads to lengthening of the polyglutamine tract in the AR protein. Recently, the effects of the polyglutamine-expanded AR have been explored in a number of cellular and animal models. Common themes include research on polyglutamine-containing nuclear inclusions and the effect of molecular chaperone overexpression on their formation. In addition, investigations have highlighted the role that abnormal transcriptional regulation, proteasome dysfunction and altered axonal transport may play in disease pathogenesis. These studies suggest a number of potential treatments for restoring neuronal function. One of the most interesting advances in SBMA research has been the creation of mouse models that recapitulate the key features of SBMA progression in men. Lowering testosterone levels in affected transgenic male mice rescued, and even reversed the polyglutamine-induced neuromuscular phenotype, indicating that manipulating androgen levels in men could be of therapeutic benefit. Although the question of why only a distinct subset of neurons is affected by polyglutamine expansion of the AR remains unsolved, future research will provide further insights into the mechanisms contributing to disease progression in SBMA.

Hormonal manipulation of benign metastasizing leiomyomas: report of two cases and review of the literature.

Benign metastasizing leiomyomas (BMLs) occur predominantly in women during reproductive years. The condition is characterized by uterine leiomyomas associated with the development, typically years later, of slow-growing metastatic lesions. The most commonly affected organs are the lungs, but BMLs have been reported in lymph nodes, deep soft tissues, mesentery, bones, the central nervous system, and the heart. In many cases, these lesions have an indolent course and are discovered rather incidentally. However, occasionally they can present with debilitating symptoms or even life-threatening complications. The presence of estrogen and progesterone receptors in these tumors supports their origin from uterine smooth muscle and, more importantly, makes them amenable to hormonal manipulation. Radical interventions, such as extensive tumor debulking and oophorectomy for hormonal control, although effective in many cases, are not always possible or desirable and carry significant morbidity. Here we present two cases of BMLs to illustrate the role of newer therapeutic agents, the estrogen receptor modulators and the aromatase inhibitors, in the hormonal manipulation of these tumors.

Cloning and characterization of an androgen receptor N-terminal-interacting protein with ubiquitin-protein ligase activity.

The androgen receptor (AR) N-terminal domain plays a critical role in androgen-responsive gene regulation. A novel AR N-terminal-interacting protein (ARNIP) was isolated using the yeast two-hybrid system and its interaction with amino acids 11-172 of the normal or corresponding region of the polyglutamine-expanded human AR confirmed by glutathione S-transferase pulldown assays. ARNIP cDNAs cloned from NSC-34 (mouse neuroblastoma/spinal cord) or PC-3 (human prostate adenocarcinoma) mRNA encoded highly homologous 30 kDa (261 amino acids) cysteine-rich proteins with a RING-H2 (C3H2C3 zinc finger) domain; this motif is highly conserved in predicted ARNIP-homologous proteins from several other species. Expression of the approximately 1.7 kb ARNIP mRNA was detected in various tissues by Northern blotting, but was highest in mouse testes, kidney and several neuronal cell lines. In addition, the human ARNIP protein was found to be encoded by nine exons spanning 32 kb on chromosome 4q21. In COS-1 cells, coexpression of ARNIP and AR did not affect AR ligand-binding kinetics, nor did ARNIP act as a coactivator or corepressor in transactivation assays. However, AR N-terminal:C-terminal interaction was reduced in the presence of ARNIP. Intriguingly, ARNIP, and in particular its RING-H2 domain, functioned as a ubiquitin-protein ligase in vitro in the presence of a specific ubiquitin-conjugating enzyme, Ubc4-1. Mutation of a single cysteine residue in the ARNIP RING-H2 domain (Cys145Ala) abolished this E3 ubiquitin ligase activity. Fluorescent protein tagging studies revealed that AR-ARNIP interaction was hormone-independent in COS-1 cells, and suggest that colocalization of both AR and ARNIP to the nucleus upon androgen addition may allow ARNIP to play a role in nuclear processes. Thus, identification of a novel AR-interacting protein with ubiquitin ligase activity will stimulate further investigation into the role of ubiquitination and the ubiquitin-proteasome system in AR-mediated cellular functions.

Cytochrome c oxidase subunit Vb interacts with human androgen receptor: a potential mechanism for neurotoxicity in spinobulbar muscular atrophy.

Spinobulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of the polyglutamine (polyGln) tract in the human androgen receptor (hAR). One mechanism by which polyGln-expanded proteins are believed to cause neuronotoxicity is through aberrant interaction(s) with, and possible sequestration of, critical cellular protein(s). Our goal was to confirm and further characterize the interaction between hAR and cytochrome c oxidase subunit Vb (COXVb), a nuclear-encoded mitochondrial protein. We initially isolated COXVb as an AR-interacting protein in a yeast two-hybrid screen to identify candidate proteins that interacted with normal and polyGln-expanded AR. Using the mammalian two-hybrid system, we confirm that COXVb interacts with normal and mutant AR and demonstrated that the COXVb-normal AR interaction is stimulated by heat shock protein 70. In addition, blue fluorescent protein-tagged AR specifically co-localized with cytoplasmic aggregates formed by green fluorescent protein-labeled polyGln-expanded AR in androgen-treated cells. Mitochondrial dysfunction may precede neuropathological findings in polyGln-expanded disorders and may thus represent an early event in neuronotoxicity. Interaction of COXVb and hAR, with subsequent sequestration of COXVb, may provide a mechanism for putative mitochondrial dysfunction in SBMA.

A G577R mutation in the human AR P box results in selective decreases in DNA binding and in partial androgen insensitivity syndrome.

We have characterized a novel mutation of the human AR, G577R, associated with partial androgen insensitivity syndrome. G577 is the first amino acid of the P box, a region crucial for the selectivity of receptor/DNA interaction. Although the equivalent amino acid in the GR (also Gly) is not involved in DNA interaction, the residue at the same position in the ER (Glu) interacts with the two central base pairs in the PuGGTCA motif. Using a panel of 16 palindromic probes that differ in these base pairs (PuGNNCA) in gel shift experiments with either the AR DNA-binding domain or the full length receptor, we observed that the G577R mutation does not induce binding to probes that are not recognized by the wild-type AR. However, binding to the four PuGNACA elements recognized by the wild-type AR was affected to different degrees, resulting in an altered selectivity of DNA response element recognition. In particular, AR-G577R did not interact with PuGGACA palindromes. Modeling of the complex between mutant AR and PuGNACA motifs indicates that the destabilizing effect of the mutation is attributable to a steric clash between the C beta of Arg at position 1 of the P box and the methyl group of the second thymine residue in the TGTTCPy arm of the palindrome. In addition, the Arg side chain can interact with G or T at the next position (PuGCACA and PuGAACA elements, respectively). The presence of C is not favorable, however, because of incompatible charges, abrogating binding to the PuGGACA element. Transactivation of several natural or synthetic promoters containing PuGGACA motifs was drastically reduced by the G577R mutation. These data suggest that androgen target genes may be differentially affected by the G577R mutation, the first natural mutation characterized that alters the selectivity of the AR/DNA interaction. This type of mutation may thus contribute to the diversity of phenotypes associated with partial androgen insensitivity syndrome.

Testosterone-mediated neuroprotection through the androgen receptor in human primary neurons.

Estrogen is an active neuroprotectant and is presently investigated as a potential therapy against Alzheimer's disease for women. To determine if male hormones could also be neuroprotective, we investigated the effect of testosterone, methyltestosterone, and epitestosterone at physiological concentrations on primary cultures of human neurons induced to undergo apoptosis by serum deprivation. Serum deprivation significantly induces neuronal apoptosis in a protracted fashion. As expected, physiological concentrations of 17-beta-estradiol and transcriptionally inactive 17-alpha-estradiol protect neurons against apoptosis. Similar to 17-beta-estradiol, physiological concentrations of testosterone are also neuroprotective. Androgen receptors are present at 8 +/- 2 fmol/mg protein in the neuron cultures. The non-aromatizable androgen, mibolerone, is also neuroprotective and aromatase inhibitor, 4-androsten-4-OL-3,17-dione, does not prevent testosterone-mediated neuroprotection. In contrast, anti-androgen, flutamide, eliminates testosterone-mediated neuroprotection. Testosterone analog, methyltestosterone, showed androgen receptor-dependent neuroprotection that was delayed in time indicating that a metabolite may be the active agent. The endogenous anti-androgen, epitestosterone, also showed a slight neuroprotective effect but not through the androgen receptor. These results indicate that androgens induce neuroprotection directly through the androgen receptor. These data suggest that androgens may also be of therapeutic value against Alzheimer's disease in aging males.

Variable expressivity and mutation databases: The androgen receptor gene mutations database.

For over 50 years genetics has presumed that variations in phenotypic expression have, for the most part, been the result of alterations in genotype. The importance and value of mutation databases has been based on the premise that the same gene or allelic variation in a specific gene that has been proven to determine a specific phenotype, will always produce the same phenotype. However, recent evidence has shown that so called "simple" Mendelian disorders or monogenic traits are often far from simple, exhibiting phenotypic variation (variable expressivity) that cannot be explained solely by a gene or allelic alteration. The AR gene mutations database now lists 25 cases where different degrees of androgen insensitivity are caused by identical mutations in the androgen receptor gene. In five of these cases the phenotypic variability is due to somatic mosaicism, that is, somatic mutations that occur in only certain cells of androgen-sensitive tissue. Recently, a number of other cases of variable expressivity have also been linked to somatic mosaicism. The impact of variable expressivity due to somatic mutations and mosaicism on mutation databases is discussed. In particular, the effect of an organism exhibiting genetic heterogeneity within its tissues, and the possibility of an organism's genotype changing over its lifetime, are considered to have important implications for mutation databases in the future.

Somatic mosaicism and variable expressivity.

For more than 50 years geneticists have assumed that variations in phenotypic expression are caused by alterations in genotype. Recent evidence shows that 'simple' mendelian disorders or monogenic traits are often far from simple, exhibiting phenotypic variation (variable expressivity) that cannot be explained entirely by a gene or allelic alteration. In certain cases of androgen insensitivity syndrome caused by identical mutations in the androgen receptor gene, phenotypic variability is caused by somatic mosaicism, that is, somatic mutations that occur only in certain androgen-sensitive cells. Recently, more than 30 other genetic conditions that exhibit variable expressivity have been linked to somatic mosaicism. Somatic mutations have also been identified in diseases such as prostate and colorectal cancer. Therefore, the concept of somatic mutations and mosaicism is likely to have far reaching consequences for genetics, in particular in areas such as genetic counseling.

Characterization of intracellular aggregates using fluorescently-tagged polyglutamine-expanded androgen receptor.

Spinal bulbar muscular atrophy (SBMA) is a classic CAG-repeat neurodegenerative disease. It is caused by expansion of a polyglutamine (polyGln) tract in the androgen receptor (AR). Recent evidence has indicated a potential role for nuclear and cytoplasmic inclusions in the pathogenesis of these diseases. We have used blue and green fluorescently-tagged AR to show that both wild-type (WT) and poly-Gln-expanded full-length AR can form aggregates and that aggregation is not related to cytotoxicity. Twenty to thirty-five percent of all cell types transfected into COS cells showed aggregation containing both amino- and carboxy-terminal fluorescent tags. The aggregates reacted with (F39.4.1), an anti-AR antibody and with IC2, an expanded polyGln tract antibody. Western analysis of protein extracts revealed little evidence of proteolysis although some cleavage of the fusion proteins was seen. The general caspase inhibitor, Z-DEVD-FMK, did not affect aggregation in either wild type or polyGln-expanded GFP-AR transfected cells. Surprisingly, addition of Mibolerone a synthetic androgen significantly decreased inclusion formation in both WT and polyGln-expanded AR-transfected cells. Overall, we show that both WT and polyGln expanded full-length AR are found in aggregates and that proteolysis is not a requirement for aggregation. Our results also suggest that toxicity is not related to intracellular aggregation of polyGln expanded AR.

The polymorphic CAG repeat of the androgen receptor gene: a potential role in breast cancer in women over 40.

Previous investigations into the relationship of CAG-repeat lengths in the androgen receptor (AR) gene to female breast cancer (BC) have yielded somewhat confusing results. Decreased AR transactivational activity lowers androgen:estrogen balance, and may thereby effect functional hyperestrogenicity. This may promote the pathogenesis of BC. To elucidate whether longer CAG repeats of the AR gene (AR), which correlate with lower transactivational activity of the AR, are associated with BC in women over 40, we examined the distribution of CAG-repeat lengths in BC tissue from this population. The BC tissue was histologically graded as: Grade 1, well differentiated (WD); Grade 2, moderately differentiated (MD); and Grade 3, poorly-differentiated (PD). Analysis showed significant differences as compared to controls when CAG lengths greater than 21 were examined, and that alleles with > or = 26 repeats were 2.4-fold more frequent in BC samples than in constitutional samples from a normal population. A significant shift to greater CAG-repeat lengths, appeared in WD and MD tumors only. Our results give some indication as to the progression of BC by suggesting that hypotransactive ARs with long polyglutamine (polyGln) tracts may have a role in the initiation and/or progression of BC. PD tumors tended to have shorter than normal CAG-repeat lengths. In this case it is hypothesized that the ARs have now become hypertransactive, possibly coinciding with the estrogen resistance that is associated with PD tumors. Whether this shift is of germline or somatic origin was not clear, though the appearance in 14% of the BC samples of a third CAG-repeat length indicates that it may be somatic.

Interactions between androgen and estrogen receptors and the effects on their transactivational properties.

The physiological interplay of androgen and estrogen action in endocrine tissues is well recognized. The biochemical processes responsible for this interplay have yet to be fully defined. We have demonstrated that the androgen receptor (AR) and estrogen receptor-alpha (ERalpha) can interact directly using the yeast and mammalian two-hybrid systems. These interactions occurred between the C-terminal ERalpha ligand-binding domain and either the N-terminal AR transactivational domain or the full-length AR. Estrogen receptor-beta (ERbeta) did not interact with the AR. DNA cotransfection studies employing AR, ERalpha and ERbeta expression vectors and AR- or ER-reporter gene constructs were used to identify and measure potential functional effects of AR-ER interaction. Coexpression of ERalpha with AR decreased AR transactivation by 35%; coexpression of AR with ERalpha decreased ERalpha transactivation by 74%. Coexpression of AR and ERbeta did not significantly modulate AR or ERbeta transactivation. In summary, we have shown that specific domains of AR and ERalpha physically interact and have demonstrated the functional consequences of such interaction. These results may help explain the nature of the physiological interplay between androgens and estrogens.

Human androgen receptor mutation disrupts ternary interactions between ligand, receptor domains, and the coactivator TIF2 (transcription intermediary factor 2).

The androgen receptor (AR) is a ligand-dependent X-linked nuclear transcription factor regulating male sexual development and spermatogenesis. The receptor is activated when androgen binds to the C-terminal ligand-binding domain (LBD), triggering a cascade of molecular events, including interactions between the LBD and the N-terminal transactivation domain (TAD), and the recruitment of transcriptional coactivators. A nonconservative asparagine to lysine substitution in AR residue 727 was encountered in a phenotypically normal man with subfertility and depressed spermatogenesis. This N727K mutation, although located in the LBD, did not alter any ligand-binding characteristic of the AR in the patient's fibroblasts or when expressed in heterologous cells. Nonetheless, the mutant AR displayed only half of wild-type transactivation capacity when exposed to physiological or synthetic androgens. This transactivation defect was consistently present when examined with two different reporter systems in three cell lines, using three androgen-driven promoters (including the complex human prostate-specific antigen promoter), confirming the pathogenicity of the mutation. In mammalian two-hybrid assays, N727K disrupted LBD interactions with the AR TAD and with the coactivator, transcription intermediary factor 2 (TIF2). Strikingly, the transactivation defect of the mutant AR can be rectified in vitro with mesterolone, consistent with the ability of this androgen analog to restore sperm production in vivo. Mesterolone, but not the physiological androgen dihydrotestosterone, restored mutant LBD interactions with the TAD and with TIF2, when expressed as fusion proteins in the two-hybrid assay. Our data support an emerging paradigm with respect to AR mutations in the LBD and male infertility: pathogenicity is transmitted through reduced interdomain and coactivator interactions, and androgen analogs that are corrective in vitro may indicate hormonal therapy.

Increased T-type Ca2+ channel activity as a determinant of cellular toxicity in neuronal cell lines expressing polyglutamine-expanded human androgen receptors.

We have analyzed Ca2+ currents in two neuroblastoma-motor neuron hybrid cell lines that expressed normal or glutamine-expanded human androgen receptors (polyGln-expanded AR) either transiently or stably. The cell lines express a unique, low-threshold, transient type of Ca2+ current that is not affected by L-type Ca2+ channel blocker (PN 200-110), N-type Ca2+ channel blocker (omega-conotoxin GVIA) or P-type Ca2+ channel blocker (Agatoxin IVA) but is blocked by either Cd2+ or Ni2+. This pharmacological profile most closely resembles that of T-type Ca2+ channels [1-3]. Exposure to androgen had no effect on control cell lines or cells transfected with normal AR but significantly changed the steady-state activation in cells transfected with expanded AR. The observed negative shift in steady-state activation results in a large increase in the T-type Ca2+ channel window current. We suggest that Ca2+ overload due to abnormal voltage-dependence of transient Ca2+ channel activation may contribute to motor neuron toxicity in spinobulbar muscular atrophy (SBMA). This hypothesis is supported by the additional finding that, at concentrations that selectively block T-type Ca2+ channel currents, Ni2+ significantly reduced cell death in cell lines transfected with polyGln-expanded AR.

[Cardiologic diagnosis of pulmonary embolism: echocardiography]. / Diagnostica cardiologica dell'embolismo polmonare: l'ecocardiografia.

Pulmonary embolism (PE) represents the third more frequent cardiovascular disease following the acute coronary artery disease and stroke. The most important predisposing clinical condition for PE is represented by the deep-vein thrombosis. The clinical diagnosis of PE has a very low accuracy; so the clinical suspect has to be necessarily directed towards the performance of diagnostic procedures. Among the most used procedures, the echocardiography has a diagnostic role but also a prognostic one. Moreover, it offers precious informations useful to perform the most suitable treatment. The echocardiography features which suggest the presence of pulmonary embolism are: right ventricle and atrium dilatation, right ventricular hypokinesia, systolic flattening of the interventricular septum, tricuspid regurgitation, pulmonary artery dilatation, disappearance or reduction of the inspiratory collapse of the inferior vena cava and presence of eventual embolic sources. According to the involvement degree of right ventricular function, it is generally possible to identify a different survival. The subgroup of patients with moderate or severe right ventricular dysfunction shows a high in hospital and within 1 year death rate. For this reason the right ventricular dysfunction degree together with the hemodynamic stability, are the most important parameters in the therapeutic choice. If there is no right ventricular dysfunction a treatment with heparin is indicated. In presence of right ventricular dysfunction and hemodynamic instability, the thrombolytic treatment is necessary. If the patient is hemodynamically stable, a transesophageal echocardiography is recommended; in case of central thrombosis the thrombolytic therapy or surgery are needed, while if no embolic material is shown the heparin treatment is advisable.

The effect of the I1307K APC polymorphism on the clinicopathological features and natural history of breast cancer.

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

Analysis of exon 1 mutations in the androgen receptor gene.

Eleven mutations in exon 1 of the androgen receptor gene (AR) have been identified in 15 individuals with Androgen Insensitivity syndrome (AIS). Nine of the mutations yield a stop codon directly, or due to a frameshift, in individuals with complete AIS (CAIS). One individual with CAIS had three different mutations in exon 1: one is nominally silent (Glu 211; GAG 995 GAA); two are missense (Pro 390 Arg and Glu 443 Arg). Five unrelated individuals with either CAIS, partial AIS (PAIS) or mild AIS (MAIS) had GAG 995 GAA as their only alteration. This report almost doubles the number of exon 1 mutations stored in the AR Mutation Database, reinforces their highly predominant nonsense character, and identifies Pro 390 and/or Gln 443 as residues that are probably necessary for one or more specific functions of the AR's N-terminal transactivation domain.