Expression of insulin-like growth factor-II and insulin-like growth factor binding proteins during Caco-2 cell proliferation and differentiation

The components of the insulin-like growth factor (IGF) axis and their roles in regulating proliferation and differentiation of the human colon adenocarcinoma cell line, Caco-2, have been investigated. Caco-2 cells proliferated in serum-free medium at 75% the rate observed in medium containing 10% fetal bovine serum. IGF-I (10 nM) increased Caco-2 cell growth in serum-free medium, but not to the rate seen with serum. Multiple IGF-II mRNA species were produced by Caco-2 cells, but IGF-I mRNA was undetectable. Secretion of radioimmunoassayable IGF-II corresponded with steady-state levels of IGF-II mRNA, neither of which was observed to change markedly over the course of 16 days of Caco-2 cell differentiation. Levels of sucrase-isomaltase mRNA, a marker for enterocytic differentiation, increased 12-fold between days 5 and 16 of culture. Northern blotting of total RNA and ligand blot and immunoblot analyses of serum-free conditioned medium revealed that Caco-2 cells produce several IGF binding proteins (IGFBPs), including IGFBP-2, -3, and -4, as well as a 31,000 M, species that was not identified. The pattern of IGFBP secretion changed dramatically during Caco-2 cell differentiation: IGFBP-3 and IGFBP-2 increased 8.5-fold and 5-fold, respectively, whereas IGFBP-4 and the 31,000 M, species decreased 43% and 90%. Caco-2 cell clones stably transfected with a human IGFBP-4 cDNA construct exhibited a 60% increase in steady-state level of IGFBP-4 mRNA, and secreted twice as much IGFBP-4 protein as controls. Moreover, IGFBP-4-overexpressing cells proliferated at only 25% the rate of control cells in serum-free medium, in conjunction with a 70% increase in expression of sucrase-isomaltase. In summary, these studies indicate that a complex IGF axis is involved in autocrine regulation of Caco-2 cell proliferation and differentiation. © 1996 Wiley-Liss, Inc.     

Progress in understanding the biosynthesis of amylose

The storage of glucose in insoluble granules is a distinctive feature of plant cells. Biosynthesis of amylose, the minor low molecular mass fraction of starch occurs from ADP-glucose. This takes place within the polysaccharide matrix through the action of granule-bound starch synthase, the major protein associated with the granule. Recently, amylose has been successfully synthesized in vitro from purified granules. Two models have been proposed to explain the mechanism of amylose synthesis in plants. The first calls for priming of synthesis through small-size malto-oligosaccharides. The second suggests that glucans are extended by granule-bound starch synthase from a high molecular mass primer present within the granule. This extension is terminated through cleavage to produce amylose. This process is subsequently repeated to give several rounds of amylose synthesis.     

Der3p/Hrd1p Is Required for Endoplasmic Reticulum-associated Degradation of Misfolded Lumenal and Integral Membrane Proteins

We have studied components of the endoplasmic reticulum (ER) proofreading and degradation system in the yeast Saccharomyces cerevisiae. Using a der3–1 mutant defective in the degradation of a mutated lumenal protein, carboxypeptidase yscY (CPY*), a gene was cloned which encodes a 64-kDa protein of the ER membrane. Der3p was found to be identical with Hrd1p, a protein identified to be necessary for degradation of HMG-CoA reductase. Der3p contains five putative transmembrane domains and a long hydrophilic C-terminal tail containing a RING-H2 finger domain which is oriented to the ER lumen. Deletion of DER3 leads to an accumulation of CPY* inside the ER due to a complete block of its degradation. In addition, a DER3 null mutant allele suppresses the temperature-dependent growth phenotype of a mutant carrying the sec61–2 allele. This is accompanied by the stabilization of the Sec61–2 mutant protein. In contrast, overproduction of Der3p is lethal in a sec61–2 strain at the permissive temperature of 25°C. A mutant Der3p lacking 114 amino acids of the lumenal tail including the RING-H2 finger domain is unable to mediate degradation of CPY* and Sec61–2p. We propose that Der3p acts prior to retrograde transport of ER membrane and lumenal proteins to the cytoplasm where they are subject to degradation via the ubiquitin-proteasome system. Interestingly, in ubc6-ubc7 double mutants, CPY* accumulates in the ER, indicating the necessity of an intact cytoplasmic proteolysis machinery for retrograde transport of CPY*. Der3p might serve as a component programming the translocon for retrograde transport of ER proteins, or it might be involved in recognition through its lumenal RING-H2 motif of proteins of the ER that are destined for degradation.     

Direct Measurement of Calcium Transport across Chloroplast Inner-Envelope Vesicles

The initial rate of Ca²⁺ movement across the inner-envelope membrane of pea (Pisum sativum L.) chloroplasts was directly measured by stopped-flow spectrofluorometry using membrane vesicles loaded with the Ca²⁺-sensitive fluorophore fura-2. Calibration of fura-2 fluorescence was achieved by combining a ratiometric method with Ca²⁺-selective minielectrodes to determine pCa values. The initial rate of Ca²⁺ influx in predominantly right-side-out inner-envelope membrane vesicles was greater than that in largely inside-out vesicles. Ca²⁺ movement was stimulated by an inwardly directed electrochemical proton gradient across the membrane vesicles, an effect that was diminished by the addition of valinomycin in the presence of K⁺. In addition, Ca²⁺ was shown to move across the membrane vesicles in the presence of a K⁺ diffusion potential gradient. The potential-stimulated rate of Ca²⁺ transport was slightly inhibited by diltiazem and greatly inhibited by ruthenium red. Other pharmacological agents such as LaCl₃, verapamil, and nifedipine had little or no effect. These results indicate that Ca²⁺ transport across the chloroplast inner envelope can occur by a potential-stimulated uniport mechanism.     

Lipoprotein lipase activity in white adipose tissue of rats subjected to exercise--rest cycles

This study was conducted to determine serum lipid levels and the activity of lipoprotein lipase in epididymal white adipose tissue of rats undergoing exercise training. During the 8-week period of treatment, one group of rats was kept sedentary and the remaining animals were exercise trained either continually (1 h of daily treadmill running) or intermittently (alternate weeks of daily running and inactivity). Exercise training, either continual or intermittent, decreased postprandial serum total and high-density lipoprotein cholesterol concentrations, which returned to sedentary levels in the intermittently trained animals following a week of rest. Lipoprotein lipase activity in whole epididymal adipose pad was lower in rats trained continually than in the sedentary group at the end of the treatment. The intermittent training program elicited large fluctuations in both the specific (per milligram of protein) and total (per tissue) activity of lipoprotein lipase in white adipose tissue. During rest periods, enzyme activity rose to levels that were higher than those of sedentary rats, whereas lipase activity was below that of sedentary animals following a week of running. In the last exercise--rest cycle, body weight gain of the intermittently trained rats was nearly abolished during the week of running, but it increased above that of sedentary animals during weeks of rest. The present results suggest that the modulation of lipoprotein lipase activity in white adipose tissue is one of the adaptations that take place to accommodate the fluctuations in the rate of energy deposition that occur in the rat during an intermittent training program.     

Discrimination of jittered sonar echoes by the echolocating bat,Eptesicus fuscus: The shape of target images in echolocation

1. Behavioral experiments with jittering echoes examined acoustic images of sonar targets in the echolocating bat, Eptesicus fuscus, along the echo delay or target range axis. Echo phase, amplitude, bandwidth, and signal-to-noise ratio were manipulated to assess the underlying auditory processes for image formation. 2. Fine delay acuity is about 10 ns. Calibration and control procedures indicate that this represents temporal acuity rather than spectral discrimination. Jitter discrimination curves change in phase when the phase of one jittering echo is shifted by 180 degrees relative to the other, showing that echo phase is involved in delay estimation. At an echo detectability index of about 36 dB, fine acuity is 40 ns, which is approximately as predicted for the delay accuracy of an ideal receiver. 3. Compound performance curves for 0 degrees and 180 degrees phase conditions match the crosscorrelation function of the echoes. The locations of both 0 degrees and 180 degrees phase peaks in the performance curves shift along the time axis by an amount that matches neural amplitude-latency trading in Eptesicus, confirming a temporal basis for jitter discrimination.     

Epigenetic regulation of neuronal dendrite and dendritic spine development

Dendrites and the dendritic spines of neurons play key roles in the connectivity of the brain and have been recognized as the locus of long-term synaptic plasticity, which is correlated with learning and memory. The development of dendrites and spines in the mammalian central nervous system is a complex process that requires specific molecular events over a period of time. It has been shown that specific molecules are needed not only at the spine’s point of contact, but also at a distance, providing signals that initiate a cascade of events leading to synapse formation. The specific molecules that act to signal neuronal differentiation, dendritic morphology, and synaptogenesis are tightly regulated by genetic and epigenetic programs. It has been shown that the dendritic spine structure and distribution are altered in many diseases, including many forms of mental retardation (MR), and can also be potentiated by neuronal activities and an enriched environment. Because dendritic spine pathologies are found in many types of MR, it has been proposed that an inability to form normal spines leads to the cognitive and motor deficits that are characteristic of MR. Epigenetic mechanisms, including DNA methylation, chromatin remodeling, and the noncoding RNA-mediated process, have profound regulatory roles in mammalian gene expression. The study of epigenetics focuses on cellular effects that result in a heritable pattern of gene expression without changes to genomic encoding. Despite extensive efforts to understand the molecular regulation of dendrite and spine development, epigenetic mechanisms have only recently been considered. In this review, we will focus on epigenetic mechanisms that regulate the development and maturation of dendrites and spines. We will discuss how epigenetic alterations could result in spine abnormalities that lead to MR, such as is seen in fragile X and Rett syndromes. We will also discuss both general methodology and recent technological advances in the study of neuronal dendrites and spines.     

Association of polyaminergic loci with anxiety, mood disorders, and attempted suicide

Background

The polyamine system has been implicated in a number of psychiatric conditions, which display both alterations in polyamine levels and altered expression of genes related to polyamine metabolism. Studies have identified associations between genetic variants in spermidine/spermine N1-acetyltransferase (SAT1) and both anxiety and suicide, and several polymorphisms appear to play important roles in determining gene expression.

Methodology/Principal Findings

We genotyped 63 polymorphisms, spread across four polyaminergic genes (SAT1, spermine synthase (SMS), spermine oxidase (SMOX), and ornithine aminotransferase like-1 (OATL1)), in 1255 French-Canadian individuals who have been followed longitudinally for 22 years. We assessed univariate associations with anxiety, mood disorders, and attempted suicide, as assessed during early adulthood. We also investigated the involvement of gene-environment interactions in terms of childhood abuse, and assessed internalizing and externalizing symptoms as endophenotypes mediating these interactions. Overall, each gene was associated with at least one main outcome: anxiety (SAT1, SMS), mood disorders (SAT1, SMOX), and suicide attempts (SAT1, OATL1). Several SAT1 polymorphisms displayed disease-specific risk alleles, and polymorphisms in this gene were involved in gene-gene interactions with SMS to confer risk for anxiety disorders, as well as gene-environment interactions between childhood physical abuse and mood disorders. Externalizing behaviors demonstrated significant mediation with regards to the association between OATL1 and attempted suicide, however there was no evidence that externalizing or internalizing behaviors were appropriate endophenotypes to explain the associations with mood or anxiety disorders. Finally, childhood sexual abuse did not demonstrate mediating influences on any of our outcomes.

Conclusions/Significance

These results demonstrate that genetic variants in polyaminergic genes are associated with psychiatric conditions, each of which involves a set of separate and distinct risk alleles. As several of these polymorphisms are associated with gene expression, these findings may provide mechanisms to explain the alterations in polyamine metabolism which have been observed in psychiatric disorders.     

Unifying Charge Generation,Recombination, and Extraction in Low‐Offset Non‐Fullerene Acceptor Organic Solar Cells

Even though significant breakthroughs with over 18% power conversion efficiencies (PCEs) in polymer:non‐fullerene acceptor (NFA) bulk heterojunction organic solar cells (OSCs) have been achieved, not many studies have focused on acquiring a comprehensive understanding of the underlying mechanisms governing these systems. This is because it can be challenging to delineate device photophysics in polymer:NFA blends comprehensively, and even more complicated to trace the origins of the differences in device photophysics to the subtle differences in energetics and morphology. Here, a systematic study of a series of polymer:NFA blends is conducted to unify and correlate the cumulative effects of i) voltage losses, ii) charge generation efficiencies, iii) non‐geminate recombination and extraction dynamics, and iv) nuanced morphological differences with device performances. Most importantly, a deconvolution of the major loss processes in polymer:NFA blends and their connections to the complex BHJ morphology and energetics are established. An extension to advanced morphological techniques, such as solid‐state NMR (for atomic level insights on the local ordering and donor:acceptor π? π interactions) and resonant soft X‐ray scattering (for donor and acceptor interfacial area and domain spacings), provide detailed insights on how efficient charge generation, transport, and extraction processes can outweigh increased voltage losses to yield high PCEs.