Targeted ablation of gonadotrophs in transgenic mice depresses prolactin but not growth hormone gene expression at birth as measured by quantitative mRNA detection

We previously reported that transgenic ablation of gonadotrophs results in impaired development of cells immunostainable for prolactin (PRL) but not of cells immunostainable for growth hormone (GH) or proopiomelanocortin (POMC) in pituitary of newborn mice. The question remained whether this reduction in PRL protein is a reflection of reduced PRL mRNA expression, or whether this regulation is only situated at the translational level. We therefore generated a new series of transgenic mice in which gonadotrophs were ablated by diphtheria toxin A targeting, and analyzed hormone mRNA levels instead of hormone protein around the day of birth. Pituitary mRNA expression levels of luteinizing hormone- (LH), PRL and GH were quantified using real-time TaqMan RT-PCR. Of the 13 transgenic mice obtained, 8 showed a clear-cut reduction (ranging from 62 to 98%) in LH mRNA levels. PRL mRNA values were significantly reduced in the transgenic mice (p=0.0034), while GH mRNA expression was unaffected (p=0.93). An additional observation was that female newborn mice produce 5 times more LH mRNA than male mice whereas no sex difference was observed for expression levels of PRL and GH mRNA. Moreover, in the wild-type mice, LH mRNA expression was 20-fold higher than GH mRNA expression which in turn was 500- to 1,000-fold higher than PRL mRNA expression, suggesting a low expression level of the PRL gene at birth. In conclusion, the present data support the hypothesis that embryonic development of PRL gene expression is stimulated by gonadotrophs.     

Mass spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab, Cancer borealis

The crustacean stomatogastric ganglion (STG) is modulated by both locally released neuroactive compounds and circulating hormones. This study presents mass spectrometric characterization of the complement of peptide hormones present in one of the major neurosecretory structures, the pericardial organs (POs), and the detection of neurohormones released from the POs. Direct peptide profiling of Cancer borealis PO tissues using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) revealed many previously identified peptides, including proctolin, red pigment concentrating hormone (RPCH), crustacean cardioactive peptide (CCAP), several orcokinins, and SDRNFLRFamide. This technique also detected corazonin, a well-known insect hormone, in the POs for the first time. However, most mass spectral peaks did not correspond to previously known peptides. To characterize and identify these novel peptides, we performed MALDI postsource decay (PSD) and electrospray ionization (ESI) MS/MS de novo sequencing of peptides fractionated from PO extracts. We characterized a truncated form of previously identified TNRNFLRFamide, NRNFLRFamide. In addition, we sequenced five other novel peptides sharing a common C-terminus of RYamide from the PO tissue extracts. High K+ depolarization of isolated POs released many peptides present in this tissue, including several of the novel peptides sequenced in the current study.     

Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins

Bordetella pertussis establishes infection by attaching to epithelial cells of the respiratory tract. One of its adhesins is filamentous haemagglutinin (FHA), a 500-A-long secreted protein that is rich in beta-structure and contains two regions, R1 and R2, of tandem 19-residue repeats. Two models have been proposed in which the central shaft is (i) a hairpin made up of a pairing of two long antiparallel beta-sheets; or (ii) a beta-helix in which the polypeptide chain is coiled to form three long parallel beta-sheets. We have analysed a truncated variant of FHA by electron microscopy (negative staining, shadowing and scanning transmission electron microscopy of unstained specimens): these observations support the latter model. Further support comes from detailed sequence analysis and molecular modelling studies. We applied a profile search method to the sequences adjacent to and between R1 and R2 and found additional "covert" copies of the same motifs that may be recognized in overt form in the R1 and R2 sequence repeats. Their total number is sufficient to support the tenet of the beta-helix model that the shaft domain--a 350 A rod--should consist of a continuous run of these motifs, apart from loop inserts. The N-terminus, which does not contain such repeats, was found to be weakly homologous to cyclodextrin transferase, a protein of known immunoglobulin-like structure. Drawing on crystal structures of known beta-helical proteins, we developed structural models of the coil motifs putatively formed by the R1 and R2 repeats. Finally, we applied the same profile search method to the sequence database and found several other proteins--all large secreted proteins of bacterial provenance--that have similar repeats and probably also similar structures.     

Origins of domestication and polyploidy in oca (Oxalis Tuberosa: Oxalidaceae). 2. Chloroplast-expressed glutamine synthetase data

In continuing study of the origins of the octoploid tuber crop oca, Oxalis tuberosa Molina, we used phylogenetic analysis of DNA sequences of the chloroplast-active (nuclear encoded) isozyme of glutamine synthetase (ncpGS) from cultivated oca, its allies in the "Oxalis tuberosa alliance," and other Andean Oxalis. Multiple ncpGS sequences found within individuals of both the cultigen and a yet unnamed wild tuber-bearing taxon of Bolivia were separated by molecular cloning, but some cloned sequences appeared to be artifacts of polymerase chain reaction (PCR) recombination and/or Taq error. Nonetheless, three classes of nonrecombinant sequences each joined a different part of the O. tuberosa alliance clade on the ncpGS gene tree. Octoploid oca shares two sequence classes with the Bolivian tuber-bearing taxon (of unknown ploidy level). Fixed heterozygosity of these two sequence classes in all ocas sampled suggests that they represent homeologous loci and that oca is allopolyploid. A third sequence class, found in eight of nine oca plants sampled, might represent a third homeologous locus, suggesting that oca may be autoallopolyploid, and is shared with another wild tuber-bearing species, tetraploid O. picchensis of southern Peru. Thus, ncpGS data identify these two taxa as the best candidates as progenitors of cultivated oca.     

Immediate effects of an 8-h advance shift of the rest-activity cycle on 24-h profiles of cortisol

To investigate the adaptation of plasma cortisol profiles to an abrupt phase advance of the rest-activity cycle, eight normal young subjects were submitted in a sleep laboratory to an 8-h advance shift of their sleep-wake and dark-light cycles. The shift was achieved by advancing bedtimes from 2300-0700 to 1500-2300. Blood samples were obtained at 20-min intervals for 68 consecutive hours. The shift resulted within 6-9 h in a 3- to 4-h advance of timings of the nadir of the cortisol profile and of the end of the quiescent period but had no immediate effect on the timing of cortisol acrophase. The quiescent period of cortisol secretion was shortened and fragmented. Thus a major advance shift achieved without enforcing sleep deprivation results in a rapid partial adaptation of the temporal profiles of cortisol but also in a marked disruption of the cortisol quiescent period. Sleep onset was consistently followed by a decrease in cortisol concentrations. Conversely, both sleep-wake and dark-light transitions were consistently associated with cortisol secretory pulses.     

Retroviral overexpression of bcl-2 in the embryonic chick lens influences denucleation in differentiating lens fiber cells

During the course of their differentiation, embryonic lens fibers undergo loss of their cytoplasmic organelles and nuclei. The denucleation process bears similarities to the nuclear breakdown that occurs during apoptosis. This has given rise to the hypothesis that this denucleation is analogous to apoptosis, but without the plasma membrane changes characteristic of apoptotic cell death. Previous work has shown that several members of the apoptotic cascade are active during denucleation. Here, we have overexpressed the anti-apoptotic molecule bcl-2 in developing lenses of the 8-day-old chick embryo in ovo, using the replication-competent retrovirus RCAS. We find that lenses overexpressing bcl-2 show varying degrees of distortion in comparison with untreated and negative insert controls, including a more spherical shape and disorganized fiber cells. All overexpressing lenses showed significantly higher numbers of smaller nuclei in the lens core, where denucleation begins. There was no change in cell size or pattern of proliferation. These in vivo results were confirmed in vitro using lens epithelial cell cultures, which differentiate into lentoids. The lentoids in treated cultures showed the same effect on nuclear number and size. We further found that in lenses overexpressing bcl-2 there was a reduction in the activation of caspase-9 and the cleavage of the caspase substrate DFF45, and, in the lens core, a failure of the nuclear chromatin to condense. These results provide strong support for the view that embryonic lens fiber cell denucleation is analogous to the nuclear degradation that occurs during apoptosis, and that similar control pathways are involved in both these phenomena.     

First-In-Human,Double-Blind,Placebo-Controlled,Randomized, Dose-Escalation Study of BG00010, a Glial Cell Line-Derived Neurotrophic Factor Family Member,in Subjects with Unilateral Sciatica

ObjectiveTo evaluate the safety, tolerability, and pharmacokinetics of single doses of BG00010 (neublastin, artemin, enovin) in subjects with unilateral sciatica.MethodsThis was a single-center, blinded, placebo-controlled, randomized Phase 1 sequential-cohort, dose-escalation study (ClinicalTrials.gov identifier {"type":"clinical-trial","attrs":{"text":"NCT00961766","term_id":"NCT00961766"}}NCT00961766; funded by Biogen Idec). Adults with unilateral sciatica were enrolled at The Royal Adelaide Hospital, Australia. Four subjects were assigned to each of eleven cohorts (intravenous BG00010 0.3, 1, 3, 10, 25, 50, 100, 200, 400, or 800 μg/kg, or subcutaneous BG00010 50 μg/kg) and were randomized 3:1 to receive a single dose of BG00010 or placebo. The primary safety and tolerability assessments were: adverse events; clinical laboratory parameters and vital signs; pain as measured by a Likert rating scale; intra-epidermal nerve fiber density; and longitudinal assessment of quantitative sensory test parameters. Blood, serum, and plasma samples were collected for pharmacokinetic and pharmacodynamic assessments. Subjects were blinded to treatment assignment throughout the study. The investigator was blinded to treatment assignment until the Data Safety Review Committee review of unblinded data, which occurred after day 28.ResultsBeyond the planned enrollment of 44 subjects, four additional subjects were enrolled into to the intravenous BG00010 200 μg/kg cohort after one original subject experienced mild generalized pruritus. Therefore, a total of 48 subjects were enrolled between August 2009 and December 2011; all were included in the safety analyses. BG00010 was generally well tolerated: in primary analyses, the most common treatment-emergent adverse events were changes in temperature perception, pruritus, rash, or headache; no trends were observed in clinical laboratory parameters, vital signs, intra-epidermal nerve fiber density, or quantitative sensory testing. BG00010 was not associated with any clear, dose-dependent trends in Likert pain scores. BG00010 was rapidly distributed, with a prolonged terminal elimination phase.ConclusionsThese data support the development of BG00010 for the treatment of neuropathic pain.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00961766","term_id":"NCT00961766"}}NCT00961766     

Understanding Brains: Details,Intuition, and Big Data

Understanding how the brain works requires a delicate balance between the appreciation of the importance of a multitude of biological details and the ability to see beyond those details to general principles. As technological innovations vastly increase the amount of data we collect, the importance of intuition into how to analyze and treat these data may, paradoxically, become more important.

This Essay is part of the "Where Next?" Series.

Experimental biologists collect details. In the early days, naturalists prowled their backyards, local forests, and meadows. They traveled the Amazon River and African savannahs and collected species and categorized them. These collectors of beetles and ferns then tried to formulate hypotheses about evolutionary relationships by looking at commonalities of structure, function, and development. In those days, there was an implicit belief that the passionate acquisition of detailed information about the idiosyncrasies of individual species contained the route to understanding the general principles of life. Although today’s experimental neuroscientists employ much more sophisticated methods, most retain a deep conviction that the specific properties of molecules, synapses, neurons, circuits, and connectomes are important for understanding how brains, be they small or large, work.Modern neuroscience traces much of its history to prescient physiologists, pharmacologists, and anatomists. Early anatomists such as Ramón y Cajal pioneered the use of stains to reveal the structure of neurons and to make astonishing leaps of intuition about the structure and function of brain circuits [1]. Early physiologists and pharmacologists deduced the existence of receptors and kinetics from bioassays [2,3]. Observation and reasoning from first principles led T. Graham Brown [4,5] to first articulate that reciprocal inhibition in the spinal cord could underlie the generation of rhythmic movements. Cajal and Brown anticipated systems neuroscience as we know it today: understanding how the particular properties of neurons and their connections give rise to the complex and adaptive responses that allow animals to interact with each other and their worlds.     

Critical test of some computational methods for prediction of NMR <Superscript>1</Superscript>H and <Superscript>13</Superscript>C chemical shifts

Performance of 18 DFT functionals (B1B95, B3LYP, B3PW91, B97D, BHandHLYP, BMK, CAM-B3LYP, HSEh1PBE, M06-L, mPW1PW91, O3LYP, OLYP, OPBE, PBE1PBE, tHCTHhyb, TPSSh, wB97xD, VSXC) in combinations with six basis sets (cc-pVDZ, aug-cc-pVDZ, cc-pVTZ, aug-cc-pVTZ, IGLO-II, and IGLO-III) and three methods for calculating magnetic shieldings (GIAO, CSGT, IGAIM) was tested for predicting ¹H and ¹³C chemical shifts for 25 organic compounds, for altogether 86 H and 88 C atoms. Proton shifts varied between 1.03 ppm to 12.00 ppm and carbon shifts between 7.87 ppm to 209.28 ppm. It was found that the best method for calculating ¹³C shifts is PBE1PBE/aug-cc-pVDZ with CSGT or IGAIM approaches (mae?=?1.66 ppm), for ¹H the best results were obtained with HSEh1PBE, mPW1PW91, PBE1PBE, CAM-B3LYP, and B3PW91 functionals with cc-pVTZ basis set and with CSGT or IGAIM approaches (mae?=?0.28 ppm). We found that often larger basis sets do not give better results for chemical shifts. The best basis sets for calculating ¹H and ¹³C chemical shifts were cc-pVTZ and aug-cc-pVDZ, respectively. CSGT and IGAIM NMR approaches can perform really well and are in most cases better than popular GIAO approach.

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Graphical Abstract Mean absolute errors for ¹H and ¹³C chemical shifts and computational times of neutral toluene molecule with aug-cc-pVDZ basis set and CSGT approach