Differences in Time to Disease Progression Do Not Predict for Cancer-specific Survival in Patients Receiving Immediate or Deferred Androgen-deprivation Therapy for Prostate Cancer: Final Results of EORTC Randomized Trial 30891 with 12 Years of Follow-up

BACKGROUND Trials assessing the benefit of immediate androgen-deprivation therapy (ADT) for treating prostate cancer (PCa) have often done so based on differences in detectable prostate-specific antigen (PSA) relapse or metastatic disease rates at a specific time after randomization. OBJECTIVE Based on the long-term results of European Organization for Research and Treatment of Cancer (EORTC) trial 30891, we questioned if differences in time to progression predict for survival differences. DESIGN, SETTING, AND PARTICIPANTS EORTC trial 30891 compared immediate ADT (n=492) with orchiectomy or luteinizing hormone-releasing hormone analog with deferred ADT (n=493) initiated upon symptomatic disease progression or life-threatening complications in randomly assigned T0-4 N0-2 M0 PCa patients. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Time to first objective progression (documented metastases, ureteric obstruction, not PSA rise) and time to objective castration-resistant progressive disease were compared as well as PCa mortality and overall survival. RESULTS AND LIMITATIONS After a median of 12.8 yr, 769 of the 985 patients had died (78%), 269 of PCa (27%). For patients receiving deferred ADT, the overall treatment time was 31% of that for patients on immediate ADT. Deferred ADT was significantly worse than immediate ADT for time to first objective disease progression (p<0.0001; 10-yr progression rates 42% vs 30%). However, time to objective castration-resistant disease after deferred ADT did not differ significantly (p=0.42) from that after immediate ADT. In addition, PCa mortality did not differ significantly, except in patients with aggressive PCa resulting in death within 3-5 yr after diagnosis. Deferred ADT was inferior to immediate ADT in terms of overall survival (hazard ratio: 1.21; 95% confidence interval, 1.05-1.39; p [noninferiority]=0.72, p [difference] = 0.0085). CONCLUSIONS This study shows that if hormonal manipulation is used at different times during the disease course, differences in time to first disease progression cannot predict differences in disease-specific survival. A deferred ADT policy may substantially reduce the time on treatment, but it is not suitable for patients with rapidly progressing disease.

Congenital proprotein convertase 1/3 deficiency causes malabsorptive diarrhea and other endocrinopathies in a pediatric cohort

BACKGROUND & AIMS Proprotein convertase 1/3 (PC1/3) deficiency, an autosomal-recessive disorder caused by rare mutations in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, has been associated with obesity, severe malabsorptive diarrhea, and certain endocrine abnormalities. Common variants in PCSK1 also have been associated with obesity in heterozygotes in several population-based studies. PC1/3 is an endoprotease that processes many prohormones expressed in endocrine and neuronal cells. We investigated clinical and molecular features of PC1/3 deficiency. METHODS We studied the clinical features of 13 children with PC1/3 deficiency and performed sequence analysis of PCSK1. We measured enzymatic activity of recombinant PC1/3 proteins. RESULTS We identified a pattern of endocrinopathies that develop in an age-dependent manner. Eight of the mutations had severe biochemical consequences in vitro. Neonates had severe malabsorptive diarrhea and failure to thrive, required prolonged parenteral nutrition support, and had high mortality. Additional endocrine abnormalities developed as the disease progressed, including diabetes insipidus, growth hormone deficiency, primary hypogonadism, adrenal insufficiency, and hypothyroidism. We identified growth hormone deficiency, central diabetes insipidus, and male hypogonadism as new features of PCSK1 insufficiency. Interestingly, despite early growth abnormalities, moderate obesity, associated with severe polyphagia, generally appears. CONCLUSIONS In a study of 13 children with PC1/3 deficiency caused by disruption of PCSK1, failure of enteroendocrine cells to produce functional hormones resulted in generalized malabsorption. These findings indicate that PC1/3 is involved in the processing of one or more enteric hormones that are required for nutrient absorption.

Modulatory effects of bag cell peptides in {\it Aplysia}: Behavioral and electrophysiological studies

Reproductive hormones have effects on the nervous system not directly related to reproductive function. In the rat, for example, luteinizing hormone releasing hormone has dramatic effects on learning and memory. The present work attempts to examine the effects of reproductive hormones on non-reproductive behaviors and the neural loci and mechanisms underlying these effects in Aplysia, an animal whose behaviors, reproductive hormones and neural circuitry have been well characterized.^ In Aplysia, the neurosecretory bag cells release several peptides that are responsible for eliciting egg laying. The effects of these peptides on the defensive tail-siphon withdrawal reflex, as well as sensitization of this reflex, were examined. Sensitization, a simple form of nonassociative learning, refers to the behavioral enhancement of a response to a test stimulus after the presentation of a strong stimulus, that may last minutes (short-term) or days (long-term). An extract of the bag cells (BCE) inhibited the baseline siphon component of the tail-siphon withdrawal reflex and suppressed long-term, but not short-term, sensitization of the reflex. Preliminary experiments suggest that BCE also affects the tail component of the tail-siphon withdrawal reflex.^ To determine the neural mechanisms underlying the inhibition of the baseline reflex, electrophysiological studies were performed using an in vitro analogue of the tail-siphon withdrawal reflex to examine the ability of BCE, as well as the individual bag cell peptides (BCPs), to modulate the circuitry of the reflex. Bag cell extract attenuated the synaptic strength of the monosynaptic connections between tail sensory neurons and tail motor neurons. When individually applied only $\beta$-BCP produced a similar attenuation. This effect of $\beta$-BCP was not dependent on changes in duration of the presynaptic action potential.^ An in vitro analogue of long-term sensitization training was developed to examine the mechanisms by which the BCPs may affect long-term sensitization of the tail-siphon withdrawal reflex. This analogue exhibited both short- and long-term facilitation of the connections between the tail sensory and motor neurons.^ The results of these behavioral and electrophysiological experiments suggest that the BCPs inhibit the tail-siphon withdrawal reflex, at least in part, by modulating the synaptic strength of the connections between the sensory neurons and motor neurons underlying the reflex. One candidate for this effect is $\beta$-BCP. Thus, the peptides which elicit egg laying may also serve other functions such as the inhibition of defensive reflexes. In addition, these experiments raise the possibility that BCPs may exert a long lasting effect ($>$24 hr), suppressing long-term sensitization of the tail-siphon withdrawal reflex. ^

Mechanical Loading Promoted Discogenic Differentiation of Human Mesenchymal Stem Cells Incorporated in 3D-PEG Scaffolds with RhGDF5 and RGD

Hydrogels have been described as ideal scaffolds for cells of 3D tissue constructs and hold strong promises with respect to in vitro 3D-cell-culture, where cells are isolated from native extracellular matrix (ECM). Synthesized polyethyleneglycol (PEG) hydrogels are appealing with regard to potential for cell therapy or as vehicles for drug delivery or even to regenerate tissue with similar hydrogel-like properties such as the nucleus pulposus of the intervertebral disc (IVD). Here, we tested whether incorporation of RGD motive would hinder discogenic differentiation of primary bone marrow-derived human mesenchymal stem cells (hMSCs) but favor proliferation of undifferentiated hMSCs. HMSCs were embedded in +RGD containing or without RGD PEG hydrogel and pre-conditioned with or without growth and differentiation factor-5 (rhGDF-5) for 13 days. Afterwards, all hMSCs-PEG gels were subsequently cyclically loaded (15% strain, 1Hz) for 5 consecutive days in a bioreactor to generate an IVD-like phenotype. Higher metabolic activity (resazurin assay) was found in groups with rhGDF5 in both gel types with and without RGD. Cell viability and morphology measured by confocal laser microscopy and DNA content showed decreased values (~60%) after 18 days of culture. Real-time RT-PCR of an array of 15 key genes suspected to be distinctive for IVD cells revealed moderate response to rhGDF5 and mechanical loading as also shown by histology staining. Preconditioning and mechanical loading showed relatively moderate responses revealed from both RT-PCR and histology although hMSCs were demonstrated to be potent to differentiate into chondrocyte-progenitor cells in micro- mass and 3D alginate bead culture.

Metabolic and hormonal response to intermittent high-intensity and continuous moderate intensity exercise in individuals with type 1 diabetes: a randomised crossover study.

AIMS/HYPOTHESIS To investigate exercise-related fuel metabolism in intermittent high-intensity (IHE) and continuous moderate intensity (CONT) exercise in individuals with type 1 diabetes mellitus. METHODS In a prospective randomised open-label cross-over trial twelve male individuals with well-controlled type 1 diabetes underwent a 90 min iso-energetic cycling session at 50% maximal oxygen consumption ([Formula: see text]), with (IHE) or without (CONT) interspersed 10 s sprints every 10 min without insulin adaptation. Euglycaemia was maintained using oral (13)C-labelled glucose. (13)C Magnetic resonance spectroscopy (MRS) served to quantify hepatocellular and intramyocellular glycogen. Measurements of glucose kinetics (stable isotopes), hormones and metabolites complemented the investigation. RESULTS Glucose and insulin levels were comparable between interventions. Exogenous glucose requirements during the last 30 min of exercise were significantly lower in IHE (p = 0.02). Hepatic glucose output did not differ significantly between interventions, but glucose disposal was significantly lower in IHE (p < 0.05). There was no significant difference in glycogen consumption. Growth hormone, catecholamine and lactate levels were significantly higher in IHE (p < 0.05). CONCLUSIONS/INTERPRETATION IHE in individuals with type 1 diabetes without insulin adaptation reduced exogenous glucose requirements compared with CONT. The difference was not related to increased hepatic glucose output, nor to enhanced muscle glycogen utilisation, but to decreased glucose uptake. The lower glucose disposal in IHE implies a shift towards consumption of alternative substrates. These findings indicate a high flexibility of exercise-related fuel metabolism in type 1 diabetes, and point towards a novel and potentially beneficial role of IHE in these individuals. TRIAL REGISTRATION ClinicalTrials.gov NCT02068638 FUNDING: Swiss National Science Foundation (grant number 320030_149321/) and R&A Scherbarth Foundation (Switzerland).

EXPRESSION OF THE HUMAN PLACENTAL LACTOGEN GENES

The placenta is the site of synthesis of various peptide and steroid hormones related to pregnancy. Human placental lactogen (hPL) is the predominant peptide hormone secreted by term placenta and its synthesis is tissue-specific and coupled to placenta development. The objective of this work was to study the structure and expression of the hPL.^ Poly(A('+))RNA from human term placenta was translated in a mouse-derived cell-free system. A major band corresponding to pre-hPL and a minor band comigrating with mature hPL, represent (TURN)15% of the total radioactively labeled proteins. Analysis of the poly(A('+))RNA showed a prominent band at approximately 860 nucleotides. A corresponding band was observed in Northern blots of total RNA, hybridized with {('32)P}-labeled recombinant plasmid containing a portion of hPL cDNA. Similar analyses of nuclear RNA showed at least four additional bands at 990, 1200, 1460 and 1760 nucleotides, respectively, which are likely precursors of hPL mRNA. Poly(A('+))RNA was used to construct a cDNA library, of which approximately 5% of the clones were found to hybridize to hPL DNA sequences. Heteroduplexes constructed between a clone containing a 815 bp hPL cDNA insert and a hPL genomic DNA clone revealed four small intervening sequences which can account for the lengths observed in hnRNA molecules.^ Recombinant plasmid HCS-pBR322 containing a 550 bp insert of a cDNA transcript of human placental lactogen (hPL) mRNA was ('3)H-labeled an hybridized in situ to human chromosome preparations. These experiments allowed assignment of the hPL and growth hormone (hGH) genes, which have over 90% nucleotide homology in their coding sequences, to band q22-24 of chromosome 17. A gene copy number experiment showed that both genes are present in (TURN)3 copies per haploid genome.^ Experiments were designed to determine if all members of the hPL gene cluster, consisting of four non-allelic genes, are transcribed in term placenta. Advantage was taken of differences in restriction endonuclease sites in the coding portions of the different hPL genes, to distinguish the putative cDNAs of the transcriptionally active genes. Two genes were found to be represented in the cDNA library and their cDNA transcripts were isolated and characterized. Three independent methods showed that their corresponding mRNAs are about equally represented in the hPL mRNA population. The two cDNAs code for prehPL proteins which differ at a single amino acid position. However the secreted hPLs have identical amino acid sequences. A tetramer insertion duplication was found in a palindrome area of the 3' untranslated region of one of the hPL mRNAs. ^

A Comparative Study of Development Mechanisms in Korea and Taiwan: Introductory Analysis

This article is the introduction to a special issue of The Developing Economies which presented the results of a research project by the Institute of Developing Economies that examined the development mechanisms in Korea and Taiwan. Our conclusion in this article is that their development mechanisms, despite their similar development patterns of export-led industrialization, have been essentially different: a government-led mechanism in Korea as opposed to a market-led mechanism in Taiwan. We verified this difference through comparative studies of the two economies covering trade balances, the growth of total factor productivity, the scale of enterprises and business groups, and the development processes of individual manufacturing sectors. In our explanatory discussion we propose that the difference in the mechanisms is based on: 1) the amount of accumulation in the economy at the time postwar industrialization started, 2) the relationship between government and society, and 3) the mechanism of social network formation.

Characterization of four bifunctional plant IAM/PAM-amidohydrolases capable of contributing to auxin biosynthesis.

Amidases [EC 3.5.1.4] capable of converting indole-3-acetamide (IAM) into the major plant growth hormone indole-3-acetic acid (IAA) are assumed to be involved in auxin de novo biosynthesis. With the emerging amount of genomics data, it was possible to identify over forty proteins with substantial homology to the already characterized amidases from Arabidopsis and tobacco. The observed high conservation of amidase-like proteins throughout the plant kingdom may suggest an important role of theses enzymes in plant development. Here, we report cloning and functional analysis of four, thus far, uncharacterized plant amidases from Oryza sativa, Sorghum bicolor, Medicago truncatula, and Populus trichocarpa. Intriguingly, we were able to demonstrate that the examined amidases are also capable of converting phenyl-2-acetamide (PAM) into phenyl-2-acetic acid (PAA), an auxin endogenous to several plant species including Arabidopsis. Furthermore, we compared the subcellular localization of the enzymes to that of Arabidopsis AMI1, providing further evidence for similar enzymatic functions. Our results point to the presence of a presumably conserved pathway of auxin biosynthesis via IAM, as amidases, both of monocot, and dicot origins, were analyzed.

Crystal structure of the HLA-Cw3 allotype-specific killer cell inhibitory receptor KIR2DL2

Killer cell inhibitory receptors (KIR) protect class I HLAs expressing target cells from natural killer (NK) cell-mediated lysis. To understand the molecular basis of this receptor-ligand recognition, we have crystallized the extracellular ligand-binding domains of KIR2DL2, a member of the Ig superfamily receptors that recognize HLA-Cw1, 3, 7, and 8 allotypes. The structure was determined in two different crystal forms, an orthorhombic P212121 and a trigonal P3221 space group, to resolutions of 3.0 and 2.9 Å, respectively. The overall fold of this structure, like KIR2DL1, exhibits K-type Ig topology with cis-proline residues in both domains that define β-strand switching, which sets KIR apart from the C2-type hematopoietic growth hormone receptor fold. The hinge angle of KIR2DL2 is approximately 80°, 14° larger than that observed in KIR2DL1 despite the existence of conserved hydrophobic residues near the hinge region. There is also a 5° difference in the observed hinge angles in two crystal forms of 2DL2, suggesting that the interdomain hinge angle is not fixed. The putative ligand-binding site is formed by residues from several variable loops with charge distribution apparently complementary to that of HLA-C. The packing of the receptors in the orthorhombic crystal form offers an intriguing model for receptor aggregation on the cell surface.

Absence of cytokine receptor-dependent specificity in red blood cell differentiation in vivo

Erythropoietin (EPO) is required for red blood cell development, but whether EPO-specific signals directly instruct erythroid differentiation is unknown. We used a dominant system in which constitutively active variants of the EPO receptor were introduced into erythroid progenitors in mice. Chimeric receptors were constructed by replacing the cytoplasmic tail of constitutively active variants of the EPO receptor with tails of diverse cytokine receptors. Receptors linked to granulocyte or platelet production supported complete erythroid development in vitro and in vivo, as did the growth hormone receptor, a nonhematopoietic receptor. Therefore, EPOR-specific signals are not required for terminal differentiation of erythrocytes. Furthermore, we found that cellular context can influence cytokine receptor signaling.

Somatostatin agonist pasireotide promotes a physiological state resembling short-day acclimatization in the photoperiodic male Siberian hamster (Phodopus sungorus)

Peer reviewed

High pressure fosters protein refolding from aggregates at high concentrations

High hydrostatic pressures (1–2 kbar), combined with low, nondenaturing concentrations of guanidine hydrochloride (GdmHCl) foster disaggregation and refolding of denatured and aggregated human growth hormone and lysozyme, and β-lactamase inclusion bodies. One hundred percent recovery of properly folded protein can be obtained by applying pressures of 2 kbar to suspensions containing aggregates of recombinant human growth hormone (up to 8.7 mg/ml) and 0.75 M GdmHCl. Covalently crosslinked, insoluble aggregates of lysozyme could be refolded to native, functional protein at a 70% yield, independent of protein concentration up to 2 mg/ml. Inclusion bodies containing β-lactamase could be refolded at high yields of active protein, even without added GdmHCl.

Synthesis and biological activities of potent peptidomimetics selective for somatostatin receptor subtype 2

A series of nonpeptide somatostatin agonists which bind selectively and with high affinity to somatostatin receptor subtype 2 (sst2) have been synthesized. One of these compounds, L-054,522, binds to human sst2 with an apparent dissociation constant of 0.01 nM and at least 3,000-fold selectivity when evaluated against the other somatostatin receptors. L-054,522 is a full agonist based on its inhibition of forskolin-stimulated adenylate cyclase activity in Chinese hamster ovary-K1 cells stably expressing sst2. L-054,522 has a potent inhibitory effect on growth hormone release from rat primary pituitary cells and glucagon release from isolated mouse pancreatic islets. Intravenous infusion of L-054,522 to rats at 50 μg/kg per hr causes a rapid and sustained reduction in growth hormone to basal levels. The high potency and selectivity of L-054,522 for sst2 will make it a useful tool to further characterize the physiological functions of this receptor subtype.

Adaptation of the bovine spongiform encephalopathy agent to primates and comparison with Creutzfeldt– Jakob disease: Implications for human health

There is substantial scientific evidence to support the notion that bovine spongiform encephalopathy (BSE) has contaminated human beings, causing variant Creutzfeldt–Jakob disease (vCJD). This disease has raised concerns about the possibility of an iatrogenic secondary transmission to humans, because the biological properties of the primate-adapted BSE agent are unknown. We show that (i) BSE can be transmitted from primate to primate by intravenous route in 25 months, and (ii) an iatrogenic transmission of vCJD to humans could be readily recognized pathologically, whether it occurs by the central or peripheral route. Strain typing in mice demonstrates that the BSE agent adapts to macaques in the same way as it does to humans and confirms that the BSE agent is responsible for vCJD not only in the United Kingdom but also in France. The agent responsible for French iatrogenic growth hormone-linked CJD taken as a control is very different from vCJD but is similar to that found in one case of sporadic CJD and one sheep scrapie isolate. These data will be key in identifying the origin of human cases of prion disease, including accidental vCJD transmission, and could provide bases for vCJD risk assessment.

The in vivo neuromodulatory effects of the herbal medicine ginkgo biloba

Extracts of Ginkgo biloba leaves are consumed as dietary supplements to counteract chronic, age-related neurological disorders. We have applied high-density oligonucleotide microarrays to define the transcriptional effects in the cortex and hippocampus of mice whose diets were supplemented with the herbal extract. Gene expression analysis focused on the mRNAs that showed a more than 3-fold change in their expression. In the cortex, mRNAs for neuronal tyrosine/threonine phosphatase 1, and microtubule-associated τ were significantly enhanced. Hyperphosphorylated τ is the major constituent of the neurofibrillary tangles in the brains of Alzheimer's disease patients. The expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-2, calcium and chloride channels, prolactin, and growth hormone (GH), all of which are associated with brain function, were also up-regulated. In the hippocampus, only transthyretin mRNA was upregulated. Transthyretin plays a role in hormone transport in the brain and possibly a neuroprotective role by amyloid-β sequestration. This study reveals that diets supplemented with Ginkgo biloba extract have notable neuromodulatory effects in vivo and illustrates the utility of genome-wide expression monitoring to investigate the biological actions of complex extracts.