Melatonin Chimeras Alter Reproductive Development and Photorefractoriness in Siberian Hamsters

Nightly melatonin (MEL) durations > 8 h provoke gonadal regression and decreases in body mass, whereas signals < 7 h stimulate gonadal and somatic growth in male Siberian hamsters. The authors sought to determine the minimum frequency of short MEL signals sufficient to induce the long-day phenotype in several photoperiodic traits. D,L-propranolol (hereafter propranolol) injections shortened MEL signals on the night of treatment without altering MEL on the subsequent night; this permitted interpolation of short MEL signals at variable frequencies against a background of long MEL signals (chimeras). Hamsters kept in short days (10 h light/day, 10L) were injected with propranolol 6 h after dark onset for 28 consecutive weeks beginning at 30 days of age (Week 0) either every other day or once every 3, 6, or 9 days. Control animals were injected with saline or with propranolol during the light phase or were transferred to long days (16L) at Week 0. Hamsters in 16L underwent rapid gonadal development and increases in body mass and displayed summer pelage color, as did hamsters treated with propranolol every other day. Animals treated with propranolol less frequently than every other day uniformly maintained undeveloped gonads and winter-like body weights, but pelage color becameproportionately darker with increased frequency of propranolol treatments. The onset of spontaneous testicular development in 10L was unaffected by propranolol injections. After termination of injections at Week 28, testicular regression was not observed in most 10L animals that previously had undergone spontaneous testicular development; however, 40% of hamsters that had been injected with propranolol every 3rd night did manifest the winter phenotype after Week 28. In an alternating sequence, short MEL signals completely override long signals and induce the summer phenotype. Threshold frequencies differ for MEL stimulation of long-day pelage and gonadal phenotypes. The timing and development of refractoriness to MEL does not depend in any simple manner on the number of long MEL signals or on the accumulation of a reaction product produced by long, and depleted by short, MEL signals.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/67291/2/10.1177_074873098129000345.pd

Efficient Gene Targeting by Homologous Recombination in Rat Embryonic Stem Cells

The rat is the preferred experimental animal in many biological studies. With the recent derivation of authentic rat embryonic stem (ES) cells it is now feasible to apply state-of-the art genetic engineering in this species using homologous recombination. To establish whether rat ES cells are amenable to in vivo recombination, we tested targeted disruption of the hypoxanthine phosphoribosyltransferase (hprt) locus in ES cells derived from both inbred and outbred strains of rats. Targeting vectors that replace exons 7 and 8 of the hprt gene with neomycinR/thymidine kinase selection cassettes were electroporated into male Fisher F344 and Sprague Dawley rat ES cells. Approximately 2% of the G418 resistant colonies also tolerated selection with 6-thioguanine, indicating inactivation of the hprt gene. PCR and Southern blot analysis confirmed correct site-specific targeting of the hprt locus in these clones. Embryoid body and monolayer differentiation of targeted cell lines established that they retained differentiation potential following targeting and selection. This report demonstrates that gene modification via homologous recombination in rat ES cells is efficient, and should facilitate implementation of targeted, genetic manipulation in the rat

VAC14 nucleates a protein complex essential for the acute interconversion of PI3P and PI(3,5)P2 in yeast and mouse

The signalling lipid PI(3,5)P2 is generated on endosomes and regulates retrograde traffic to the trans-Golgi network. Physiological signals regulate rapid, transient changes in PI(3,5)P2 levels. Mutations that lower PI(3,5)P2 cause neurodegeneration in human patients and mice. The function of Vac14 in the regulation of PI(3,5)P2 was uncharacterized previously. Here, we predict that yeast and mammalian Vac14 are composed entirely of HEAT repeats and demonstrate that Vac14 exerts an effect as a scaffold for the PI(3,5)P2 regulatory complex by direct contact with the known regulators of PI(3,5)P2: Fig4, Fab1, Vac7 and Atg18. We also report that the mouse mutant ingls (infantile gliosis) results from a missense mutation in Vac14 that prevents the association of Vac14 with Fab1, generating a partial complex. Analysis of ingls and two additional mutants provides insight into the organization of the PI(3,5)P2 regulatory complex and indicates that Vac14 mediates three distinct mechanisms for the rapid interconversion of PI3P and PI(3,5)P2. Moreover, these studies show that the association of Fab1 with the complex is essential for viability in the mouse

Longitudinal Evaluation of an N-Ethyl-N-Nitrosourea-Created Murine Model with Normal Pressure Hydrocephalus

Normal-pressure hydrocephalus (NPH) is a neurodegenerative disorder that usually occurs late in adult life. Clinically, the cardinal features include gait disturbances, urinary incontinence, and cognitive decline.Herein we report the characterization of a novel mouse model of NPH (designated p23-ST1), created by N-ethyl-N-nitrosourea (ENU)-induced mutagenesis. The ventricular size in the brain was measured by 3-dimensional micro-magnetic resonance imaging (3D-MRI) and was found to be enlarged. Intracranial pressure was measured and was found to fall within a normal range. A histological assessment and tracer flow study revealed that the cerebral spinal fluid (CSF) pathway of p23-ST1 mice was normal without obstruction. Motor functions were assessed using a rotarod apparatus and a CatWalk gait automatic analyzer. Mutant mice showed poor rotarod performance and gait disturbances. Cognitive function was evaluated using auditory fear-conditioned responses with the mutant displaying both short- and long-term memory deficits. With an increase in urination frequency and volume, the mutant showed features of incontinence. Nissl substance staining and cell-type-specific markers were used to examine the brain pathology. These studies revealed concurrent glial activation and neuronal loss in the periventricular regions of mutant animals. In particular, chronically activated microglia were found in septal areas at a relatively young age, implying that microglial activation might contribute to the pathogenesis of NPH. These defects were transmitted in an autosomal dominant mode with reduced penetrance. Using a whole-genome scan employing 287 single-nucleotide polymorphic (SNP) markers and further refinement using six additional SNP markers and four microsatellite markers, the causative mutation was mapped to a 5.3-cM region on chromosome 4.Our results collectively demonstrate that the p23-ST1 mouse is a novel mouse model of human NPH. Clinical observations suggest that dysfunctions and alterations in the brains of patients with NPH might occur much earlier than the appearance of clinical signs. p23-ST1 mice provide a unique opportunity to characterize molecular changes and the pathogenic mechanism of NPH

A Polymorphism in the HLA-DPB1 Gene Is Associated with Susceptibility to Multiple Sclerosis

We conducted an association study across the human leukocyte antigen (HLA) complex to identify loci associated with multiple sclerosis (MS). Comparing 1927 SNPs in 1618 MS cases and 3413 controls of European ancestry, we identified seven SNPs that were independently associated with MS conditional on the others (each ). All associations were significant in an independent replication cohort of 2212 cases and 2251 controls () and were highly significant in the combined dataset (). The associated SNPs included proxies for HLA-DRB1*15:01 and HLA-DRB1*03:01, and SNPs in moderate linkage disequilibrium (LD) with HLA-A*02:01, HLA-DRB1*04:01 and HLA-DRB1*13:03. We also found a strong association with rs9277535 in the class II gene HLA-DPB1 (discovery set , replication set , combined ). HLA-DPB1 is located centromeric of the more commonly typed class II genes HLA-DRB1, -DQA1 and -DQB1. It is separated from these genes by a recombination hotspot, and the association is not affected by conditioning on genotypes at DRB1, DQA1 and DQB1. Hence rs9277535 represents an independent MS-susceptibility locus of genome-wide significance. It is correlated with the HLA-DPB1*03:01 allele, which has been implicated previously in MS in smaller studies. Further genotyping in large datasets is required to confirm and resolve this association

Benefits of a ball and chain: simple environmental enrichments improve welfare and reproductive success in farmed American mink (Neovison vison)

Can simple enrichments enhance caged mink welfare? Pilot data from 756 sub-adults spanning three colour-types (strains) identified potentially practical enrichments, and suggested beneficial effects on temperament and fur-chewing. Our main experiment started with 2032 Black mink on three farms: from each of 508 families, one juvenile male-female pair was enriched (E) with two balls and a hanging plastic chain or length of hose, while a second pair was left as a non-enriched (NE) control. At 8 months, more than half the subjects were killed for pelts, and 302 new females were recruited (half enriched: ‘late E’). Several signs of improved welfare or productivity emerged. Access to enrichment increased play in juveniles. E mink were calmer (less aggressive in temperament tests; quieter when handled; less fearful, if male), and less likely to fur-chew, although other stereotypic behaviours were not reduced. On one farm, E females had lower cortisol (inferred from faecal metabolites). E males tended to copulate for longer. E females also weaned more offspring: about 10% more juveniles per E female, primarily caused by reduced rates of barrenness (‘late E’ females also giving birth to bigger litters on one farm), effects that our data cautiously suggest were partly mediated by reduced inactivity and changes in temperament. Pelt quality seemed unaffected, but E animals had cleaner cages. In a subsidiary side-study using 368 mink of a second colour-type (‘Demis’), similar temperament effects emerged, and while E did not reduce fur-chewing or improve reproductive success in this colour-type, E animals were judged to have better pelts. Overall, simple enrichments were thus beneficial. These findings should encourage welfare improvements on fur farms (which house 60-70 million mink p.a.) and in breeding centres where endangered mustelids (e.g. black-footed ferrets) often reproduce poorly. They should also stimulate future research into more effective practical enrichments

Identification of calcium-binding proteins associated with the human sperm plasma membrane

<p>Abstract</p> <p>Background</p> <p>The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood. Here, we investigated the repertoire of calcium-regulated proteins associated with the human sperm plasma membrane.</p> <p>Methods</p> <p>Surface specific radioiodination was combined with two-dimensional gel electrophoresis, a 45Ca-overlay assay, computer assisted image analysis and mass spectrometry to identify calcium-binding proteins exposed on the human sperm surface.</p> <p>Results</p> <p>Nine acidic 45Ca-binding sperm proteins were excised from stained preparative 2D gels and identified by mass spectrometry. Five of the calcium binding proteins; HSPA2 (HSP70-1), HSPA5 (Bip), HYOU1 (ORP150), serum amyloid P-component (SAP) and protein kinase C substrate 80K-H (80K-H) were found to be accessible to Iodo-Bead catalyzed 125I-labelling on the surface of intact human sperm. Agglutination and immunofluorescence analysis confirmed that SAP is situated on the plasma membrane of intact, motile sperm as well as permeabilized cells. Western blot analysis showed increased phosphorylation of human sperm 80K-H protein following in vitro capacitation. This is the first demonstration of the 80K-H protein in a mammalian sperm.</p> <p>Conclusion</p> <p>The presence of SAP on the surface of mature sperm implies that SAP has a physiological role in reproduction, which is thought to be in the removal of spermatozoa from the female genital tract via phagocytosis. Since 80K-H is a Ca2+-sensor recently implicated in the regulation of both inositol 1,4,5-trisphosphate receptor and transient receptor potential (TRP) cation channel activities, its detection in sperm represents the first direct signaling link between PKC and store-operated calcium channels identified in human sperm.</p

Sex Differences in the Brain: A Whole Body Perspective

Most writing on sexual differentiation of the mammalian brain (including our own) considers just two organs: the gonads and the brain. This perspective, which leaves out all other body parts, misleads us in several ways. First, there is accumulating evidence that all organs are sexually differentiated, and that sex differences in peripheral organs affect the brain. We demonstrate this by reviewing examples involving sex differences in muscles, adipose tissue, the liver, immune system, gut, kidneys, bladder, and placenta that affect the nervous system and behavior. The second consequence of ignoring other organs when considering neural sex differences is that we are likely to miss the fact that some brain sex differences develop to compensate for differences in the internal environment (i.e., because male and female brains operate in different bodies, sex differences are required to make output/function more similar in the two sexes). We also consider evidence that sex differences in sensory systems cause male and female brains to perceive different information about the world; the two sexes are also perceived by the world differently and therefore exposed to differences in experience via treatment by others. Although the topic of sex differences in the brain is often seen as much more emotionally charged than studies of sex differences in other organs, the dichotomy is largely false. By putting the brain firmly back in the body, sex differences in the brain are predictable and can be more completely understood

Nothing Lasts Forever: Environmental Discourses on the Collapse of Past Societies

The study of the collapse of past societies raises many questions for the theory and practice of archaeology. Interest in collapse extends as well into the natural sciences and environmental and sustainability policy. Despite a range of approaches to collapse, the predominant paradigm is environmental collapse, which I argue obscures recognition of the dynamic role of social processes that lie at the heart of human communities. These environmental discourses, together with confusion over terminology and the concepts of collapse, have created widespread aporia about collapse and resulted in the creation of mixed messages about complex historical and social processes

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field