Daily and circadian rhythms of locomotor activity in the American lobster, Homarus americanus

It is widely accepted that American lobsters, Homarus americanus (Milne-Edwards), are nocturnally active. However, the degree to which this rhythm is expressed by different individuals and the underlying causes of lobster activity rhythms, are poorly understood. In order to address these issues we recorded daily patterns of lobster locomotion using two novel techniques. In the first, reed switch assemblies were used to monitor the distance traveled by freely moving lobsters (n=43), each fitted with a small magnet, as they walked around a 1 m diameter racetrack. The advantages of this technique included: (1) lobsters were freely moving; (2) the system could be deployed in laboratory tanks or in the field and; (3) actual distances moved were measured, not just relative activity. The second technique involved placing individual lobsters (n=10) into custom-designed running wheels. This allowed for continuous monitoring of locomotor activity for extended durations (>45 days) under normal light/dark (L/D) cycles, as well as in constant darkness (D/D) and constant light (L/L).Under ambient light conditions lobsters in the racetracks moved an average of 60.1±6.5 m/day in flow-through seawater tanks. Overall, lobsters were significantly more active at night, moving 4.1±0.4 m/h in the dark vs. 1.0±0.2 m/h in the light. However, many of the lobsters moved as much during the day as during the night.Lobsters in the running wheels moved an average of 36.6±11.7 m/day and 80% expressed clear daily rhythms of activity, with a mean periodicity of 24.0±0.1 h under L/D conditions. Under D/D conditions 90% of the animals expressed free-running circadian rhythms with a mean periodicity of 24.2±0.3 h, indicating that this species possesses endogenous rhythmicity. While the running wheel results show that the nocturnal pattern of locomotor activity for this species is strongly influenced by an endogenous circadian clock, the results from the racetracks show that there is remarkable variability in the extent to which they express this pattern under natural conditions.     

Turnover of mouse intestinal brush border membrane proteins and enzymes in organ culture: A direct evaluation from studies on the evolution of enzyme activities during the culture

The turnover of mouse intestinal brush border membrane enzymes has been studied by kinetic analysis of the evolution of enzyme activities during organ culture. By comparing the results obtained in these studies with the predictions from a mathematical model of enzyme synthesis and degradation in orgen cultures, it has been possible to reach the following conclusions: (1) There is no degradation of brush border membrane enzymes during culture and the rate of synthesis of each enzyme is directly measurable from the kinetics of total enzyme accumulation (tissue + media). (2)_Brush border membrane enzymes are released in culture media by two complementary processes. The first one involves a differential solubilization of enzymes but its exact nature cannot be exactly stated. The second one involves a microvesiculation of brush border membranes, the importance of which in vivo is seen in the possible conciliation between unitary membrane synthesis and heterogeneous turnover of membrane components.     

Receptor-independent metabolism of platelet-activating factor by myelogenous cells

Human neutrophils incorporate and metabolize platelet-activating factor (PAF). We dissociated these events from PAF binding to its receptors. Cells were pretreated with either pronase, a PAF antagonist (L652731), or excess PAF. This reduced PAF receptor numbers by 70 to almost 100% but had no comparable effect upon the neutrophil's ability to metabolize PAF. Furthermore, HL-60 cells efficiently metabolized, but did not specifically bind, PAF. Thus, PAF receptor availability did not correlate with PAF metabolic capacity and we conclude that myelogenous tissues can process this bioactive ligand by a receptor-independent pathway.     

Inventing Engineered Organoids for end-stage liver failure patients

Journal of Molecular Histology - End-stage liver disease (ESLD) is a term used clinically in reference to a group of liver diseases with liver transplantation as the choice of treatment. Due to the...     

An organelle proteomic method to study neurotransmission-related proteins, applied to a neurodevelopmental model of schizophrenia

Limited information is currently available on molecular events that underlie schizophrenia-like behaviors in animal models. Accordingly, we developed an organelle proteomic approach enabling the study of neurotransmission-related proteins in the prefrontal cortex (PFC) of postpubertal (postnatal day 60 (PD60)) neonatally ventral hippocampal (nVH) lesioned rats, an extensively used neurodevelopmental model of schizophrenia-like behaviors. The PFC was chosen because of its purported role in the etiology of the disease. Statistical analysis of 392 reproducible spots on 2-D organelle proteomic patterns revealed significant changes in intensity of 18 proteinous spots in plasma membrane-enriched fractions obtained from postpubertal nVH lesioned rats compared to controls. Mass spectrometric analysis and database searching allowed the identification of a single protein in each of the nine differential spots, including proteins of low abundance, such as neurocalcin delta. Most of the identified dysregulated proteins, including clathrin light chain B, syntaxin binding protein 1b and visinin-like protein 1 are known to be linked to various neurotransmitter systems and to play key roles in plasma membrane receptor expression and recycling as well as synaptic vesicle exocytosis/recycling. Organelle proteomic approaches have hence proved to be most useful to identify key proteins linked to a given behavior in animal models of brain diseases.     

Heterogeneous Nuclear Ribonucleoprotein K Represses the Production of Pro-apoptotic Bcl-xS Splice Isoform

The Bcl-x pre-mRNA is alternatively spliced to produce the anti-apoptotic Bcl-x_L and the pro-apoptotic Bcl-x_S isoforms. By performing deletion mutagenesis on a human Bcl-x minigene, we have identified a novel exonic element that controls the use of the 5′ splice site of Bcl-x_S. The proximal portion of this element acts as a repressor and is located downstream of an enhancer. Further mutational analysis provided a detailed topological map of the regulatory activities revealing a sharp transition between enhancer and repressor sequences. Portions of the enhancer can function when transplanted in another alternative splicing unit. Chromatography and immunoprecipitation assays indicate that the silencer element interacts with heterogeneous ribonucleoprotein particle (hnRNP) K, consistent with the presence of putative high affinity sites for this protein. Finally, down-regulation of hnRNP K by RNA interference enhanced splicing to Bcl-x_S, an effect seen only when the sequences bound by hnRNP K are present. Our results therefore document a clear role for hnRNP K in preventing the production of the pro-apoptotic Bcl-x_S splice isoform.Alternative splicing is a major mechanism used to augment the number of proteins encoded by the genome. It is estimated that as many as 97% of multiple exon pre-mRNAs undergo alternative splicing (1, 2). Disruption of alternative splicing by mutating important regulatory sequences or by altering the expression or activity of proteins controlling splice site selection has been linked with different diseases, including cancer (3–7). Apoptosis is an important and complex cellular program involved in development and differentiation in higher organisms (8, 9). However, its aberrant control often contributes to cancer development and the resistance of cancer cells to drug therapy (10–13).Genes implicated in the apoptotic pathway are alternatively spliced often to produce protein isoforms with distinct or even antagonistic activities (14, 15). A good example is the apoptotic regulator Bcl-x, which is alternatively spliced to produce two major isoforms, the anti-apoptotic Bcl-x_L protein and the shorter pro-apoptotic Bcl-x_S isoform (16). This alternative splicing decision involves a competition between two 5′ splice sites; the use of the downstream site creates Bcl-x_L, and the use of the upstream one produces Bcl-x_S (Fig. 1A). Bcl-x_L is always the predominant form in cancer cells, and overexpressing it can confer resistance to chemotherapeutic agents (17–22). On the other hand, overexpression of the pro-apoptotic Bcl-x_S isoform enhances sensitivity to the topoisomerase inhibitor etoposide and to taxol in a breast cancer cell line, while triggering apoptosis in melanoma cell lines (23, 24). Using antisense technologies to improve the production of the Bcl-x_S splice variant can also induce apoptosis in cancer cells (25–27).Open in a separate windowFIGURE 1.A, alternative splicing of Bcl-x produces two major isoforms, Bcl-x_L and Bcl-x_S. B, regulation of Bcl-x alternative splicing. The enhancer elements are shown as white boxes, and the repressors are black. The pointed and flat arrows indicate positive and negative regulation, respectively. Protein kinase C inhibition relieves repression caused by the SB1 element on the Bcl-x_S splice site (36). The repressor elements CRCE1, recognized by SAP155, and CRCE2 mediate the production of Bcl-x_S by ceramide as when induced by gemcitabine in A549 cells (38, 39). hnRNP F/H binds to the B2G element to enhance the production of the Bcl-x_S isoform (41). RBM25, through an element located upstream of the Bcl-x_S splice site, can also augment its use (44). A large intronic region (IRE) mediates the Bcl-x_L increase caused by interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and 12-O-tetradecanoylphorbol-13-acetate (TPA) (35). Finally, the B3 region also enhances Bcl-x_L formation through the binding of SRp30c to AM2 and ML2 and the U1 snRNP to two cryptic 5′ splice sites (42).Alternative splicing is regulated by different proteins bound to sequence elements near splice sites. A variety of mechanisms is used to achieve regulation. Some splicing factors act by recruiting or inhibiting the binding of different components of the spliceosome. Others may change the conformation of the pre-mRNA to mask a splice site or to bring a pair of splice sites into closer proximity (28, 29).Although individual factors can have a strong and specific effect on splicing decisions, alternative splicing often relies on a combination of factors to determine the appropriate levels of isoforms. The implication of multiple proteins likely provides additional levels of regulation that helps attuned splicing control to a variety of stresses, environmental cues, and growth conditions. In several cases, the interaction of regulatory factors can be antagonistic. For example, in the Drosophila male-specific-lethal-2 (msl-2) pre-mRNA, recruitment of SXL to a uridine-rich region interferes with the binding of TIA-1 that is necessary for efficient U1 snRNP² recruitment at the 5′ splice site (30). On the same pre-mRNA, SXL also diminishes U2AF recognition of the polypyrimidine tract at the 3′ splice site. TIA proteins bound to a U-rich element on the avian myosin phosphatase targeting subunit-1 (MYPT1) pre-mRNA repress the binding of PTB (31). PTB can also reduce the recruitment of ETR-3 to intronic elements near exon 5 of cardiac troponin T (32). In neurons, the binding of PTB to the introns surrounding the N1 exon of c-src is antagonized by nPTB protein, promoting exon inclusion. On the hnRNP A1 pre-mRNA, PTB diminishes the binding of SRp30c to the intronic CE9 element, reducing the inhibition by this protein on the use of the downstream 3′ splice site (33). SC35 and hnRNP A1 have partially overlapping binding sites on the human immunodeficiency virus 1 (HIV-1) tat exon 2. Preferential binding of SC35 enhances the inclusion of the exon, whereas hnRNP A1, by reducing SC35 binding, increases exclusion (34). Thus, the competition provided by an overlapping or a closely abutting pair of enhancer/ silencer represents a simple and frequent mechanism of splicing control.The regulation of Bcl-x alternative splicing has received some attention in recent years leading to the discovery of several cis-acting elements and a few trans-acting control factors (Fig. 1B). Intronic regions downstream from the Bcl-x_L 5′ splice site have been implicated as mediating signals from cytokines such as interleukin-6 and granulocyte-macrophage colony-stimulating factor (35). In addition, we have reported that an element located 187 nt upstream of the Bcl-x_S splice site mediates a protein kinase C-dependent signal that represses splicing to the Bcl-x_S donor site (36). On the other hand, ceramide enhances the use of the Bcl-x_S 5′ splice site by lifting the repression mediated by two other elements (37, 38). The activity of one of these apparently involves SAP155 (39). The RNA-binding protein Sam68, under the control of the tyrosine kinase Fyn, can also increase the production of Bcl-x_S in cooperation with hnRNP A1 (40), and this effect is inhibited by overexpression of ASF/SF2. The Bcl-x sequences bound by the above factors remain to be identified. We also uncovered enhancer elements for Bcl-x_S and Bcl-x_L. hnRNP F and H bind downstream of the Bcl-x_S 5′ splice site to stimulate splicing to that site (41). Enhancement of Bcl-x_L is conferred by SRp30c, which binds upstream of the 5′ splice site to antagonize the repressor activity of pseudo 5′ splice sites (42). Recently, the SR protein SC35 was shown to increase the production of Bcl-x_S (43). Finally, the binding of RBM25 to a sequence element upstream of the Bcl-x_S 5′ splice site stimulated its use, possibly by recruiting U1 snRNP through its interaction with the U1-associated protein hLuc7A (44). Thus, the region located between the two competing 5′ splice sites of Bcl-x is densely populated by splicing control elements.In this study, we have pursued our characterization of Bcl-x splicing control by examining the contribution of sequences directly upstream of the Bcl-x_S donor site. Our mutational approach identified a region containing flanking enhancer and silencer activities. The activity of the repressor portion is mediated by hnRNP K, which makes this protein an anti-apoptotic regulator.     

Identifying the neural circuitry of alcohol craving and relapse vulnerability

With no further intervention, relapse rates in detoxified alcoholics are high and usually exceed 80% of all detoxified patients. It has been suggested that stress and exposure to priming doses of alcohol and to alcohol-associated stimuli (cues) contribute to the relapse risk after detoxification. This article focuses on neuronal correlates of cue responses in detoxified alcoholics. Current brain imaging studies indicate that dysfunction of dopaminergic, glutamatergic and opioidergic neurotransmission in the brain reward system (ventral striatum including the nucleus accumbens) can be associated with alcohol craving and functional brain activation in neuronal systems that process attentional relevant stimuli, reward expectancy and experience. Increased functional brain activation elicited by such alcohol-associated cues predicted an increased relapse risk, whereas high brain activity elicited by affectively positive stimuli may represent a protective factor and was correlated with a decreased prospective relapse risk. These findings are discussed with respect to psychotherapeutic and pharmacological treatment options.