Measuring Lactase Enzymatic Activity in the Teaching Lab.

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 °C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

Prevention of long-term memory loss after retrieval by an endogenous CaMKII inhibitor.

CaMK2N1 and CaMK2N2 are endogenous inhibitors of calcium/calmodulin-dependent protein kinase II (CaMKII), a key synaptic signaling molecule for learning and memory. Here, we investigated the learning and memory function of CaMK2N1 by knocking-down its expression in dorsal hippocampus of mice. We found that reduced CaMK2N1 expression does not affect contextual fear long-term memory (LTM) formation. However, we show that it impairs maintenance of established LTM, but only if retrieval occurs. CaMK2N1 knockdown prevents a decrease of threonine-286 (T286) autophosphorylation of αCaMKII and increases GluA1 levels in hippocampal synapses after retrieval of contextual fear LTM. CaMK2N1 knockdown can also increase CaMK2N2 expression, but we show that such increased expression does not affect LTM after retrieval. We also found that substantial overexpression of CaMK2N2 in dorsal hippocampus impairs LTM formation, but not LTM maintenance, suggesting that CaMKII activity is not required for LTM storage. Taken together, we propose a specific function for CaMK2N1; enabling LTM maintenance after retrieval by inhibiting T286 autophosphorylation of αCaMKII.

Noncoding RNAs and the control of signalling via nuclear receptor regulation in health and disease.

Nuclear receptors belong to a superfamily of proteins that play central roles in human biology, orchestrating a large variety of biological functions in both health and disease. Understanding the interactions and regulatory pathways of NRs will allow development of potential therapeutic interventions for a multitude of disease processes. Non-coding RNAs have recently been discovered to have significant interactions with NR signalling pathways via a variety of biological connections. This review summarises the known interactions between ncRNAs and the NR superfamily in health, embryogenesis and a plethora of human diseases.

The utility of patient specific induced pluripotent stem cells for the modelling of Autistic Spectrum Disorders.

Until now, models of psychiatric diseases have typically been animal models. Whether they were to be used to further understand the pathophysiology of the disorder, or as drug discovery tools, animal models have been the choice of preference in mimicking psychiatric disorders in an experimental setting. While there have been cellular models, they have generally been lacking in validity. This situation is changing with the advent of patient-specific induced pluripotent stem cells (iPSCs). In this article, we give a methodological evaluation of the current state of the iPS technology with reference to our own work in generating patient-specific iPSCs for the study of autistic spectrum disorder (ASD). In addition, we will give a broader perspective on the validity of this technology and to what extent it can be expected to complement animal models of ASD in the coming years.

αCaMKII autophosphorylation controls the establishment of alcohol-induced conditioned place preference in mice.

The autophosphorylation of alpha Ca2+ /calmodulin dependent protein kinase II (αCaMKII) is important for memory formation and is becoming increasingly implicated in the development of drug addiction. Previous work suggests that αCaMKII acts via the monoaminergic systems to facilitate the establishment of alcohol drinking behaviour. The present study aims to investigate whether αCaMKII autophosphorylation deficient αCaMKII(T286A) mice show a difference in the rewarding properties of alcohol (2 g/kg, i.p.), as measured by conditioned place preference (CPP). We found that alcohol-induced CPP could be established at an accelerated rate in αCaMKII(T286A) compared to wild type (WT) mice. Hyperactivity/hyper-arousal induced by the test environment was normalised by alcohol in the αCaMKII(T286A), but not WT mice. This effect could be conditioned to the test environment and may suggest enhanced negative reinforcing action of alcohol in αCaMKII autophosphorylation deficient mice.

αCaMKII autophosphorylation controls the establishment of alcohol drinking behavior.

The α-Ca(2+)/calmodulin-dependent protein kinase II (αCaMKII) is a crucial enzyme controlling plasticity in the brain. The autophosphorylation of αCaMKII works as a 'molecular memory' for a transient calcium activation, thereby accelerating learning. We investigated the role of αCaMKII autophosphorylation in the establishment of alcohol drinking as an addiction-related behavior in mice. We found that alcohol drinking was initially diminished in αCaMKII autophosphorylation-deficient αCaMKII(T286A) mice, but could be established at wild-type level after repeated withdrawals. The locomotor activating effects of a low-dose alcohol (2 g/kg) were absent in αCaMKII(T286A) mice, whereas the sedating effects of high-dose (3.5 g/kg) were preserved after acute and subchronic administration. The in vivo microdialysis revealed that αCaMKII(T286A) mice showed no dopamine (DA) response in the nucleus accumbens to acute or subchronic alcohol administration, but enhanced serotonin (5-HT) responses in the prefrontal cortex. The attenuated DA response in αCaMKII(T286A) mice was in line with altered c-Fos activation in the ventral tegmental area after acute and subchronic alcohol administration. In order to compare findings in mice with the human condition, we tested 23 single-nucleotide polymorphisms (SNPs) in the CAMK2A gene for their association with alcohol dependence in a population of 1333 male patients with severe alcohol dependence and 939 controls. We found seven significant associations between CAMK2A SNPs and alcohol dependence, one of which in an autophosphorylation-related area of the gene. Together, our data suggest αCaMKII autophosphorylation as a facilitating mechanism in the establishment of alcohol drinking behavior with changing the DA-5-HT balance as a putative mechanism.

αCaMKII autophosphorylation controls exploratory activity to threatening novel stimuli.

Autophosphorylation of αCaMKII is regarded as a 'molecular memory' for Ca(2+) transients and a crucial mechanism in aversely, but less so in appetitively, motivated learning and memory. While there is a growing body of research implicating αCaMKII in general in behavioral responses to threat or fearful stimuli, little is known about the contribution of the autophosphorylation. The present study asked how αCaMKII autophosphorylation controls anxiety-like behavioral responses toward novel, potentially threatening stimuli. We tested homozygous and heterozygous T286A αCaMKII autophosphorylation deficient mice and wild types in a systematic series of behavioral tests. Homozygous mutants were more active in the open field test and showed reduced anxiety-related behavior in the light/dark test, but these findings were confounded by a hyperlocomotor phenotype. The analysis of elevated plus maze showed significantly reduced anxiety-related behavior in the αCaMKII autophosphorylation-deficient mice which appeared to mediate a hyperlocomotor response. An analysis of home cage behavior, where neither novel nor threatening stimuli were present, showed no differences in locomotor activity between genotypes. Increased locomotion was not observed in the novel object exploration test in the αCaMKII autophosphorylation-deficient mice, implying that hyperactivity does not occur in response to discrete novel stimuli. The present data suggest that the behavior of αCaMKII autophosphorylation-deficient mice cannot simply be described as a low anxiety phenotype. Instead it is suggested that αCaMKII autophosphorylation influences locomotor reactivity to novel environments that are potentially, but not necessarily threatening.

C-terminal region of EBNA-2 determines the superior transforming ability of type 1 Epstein-Barr virus by enhanced gene regulation of LMP-1 and CXCR7.

Type 1 Epstein-Barr virus (EBV) strains immortalize B lymphocytes in vitro much more efficiently than type 2 EBV, a difference previously mapped to the EBNA-2 locus. Here we demonstrate that the greater transforming activity of type 1 EBV correlates with a stronger and more rapid induction of the viral oncogene LMP-1 and the cell gene CXCR7 (which are both required for proliferation of EBV-LCLs) during infection of primary B cells with recombinant viruses. Surprisingly, although the major sequence differences between type 1 and type 2 EBNA-2 lie in N-terminal parts of the protein, the superior ability of type 1 EBNA-2 to induce proliferation of EBV-infected lymphoblasts is mostly determined by the C-terminus of EBNA-2. Substitution of the C-terminus of type 1 EBNA-2 into the type 2 protein is sufficient to confer a type 1 growth phenotype and type 1 expression levels of LMP-1 and CXCR7 in an EREB2.5 cell growth assay. Within this region, the RG, CR7 and TAD domains are the minimum type 1 sequences required. Sequencing the C-terminus of EBNA-2 from additional EBV isolates showed high sequence identity within type 1 isolates or within type 2 isolates, indicating that the functional differences mapped are typical of EBV type sequences. The results indicate that the C-terminus of EBNA-2 accounts for the greater ability of type 1 EBV to promote B cell proliferation, through mechanisms that include higher induction of genes (LMP-1 and CXCR7) required for proliferation and survival of EBV-LCLs.

Properties of contextual memory formed in the absence of αCaMKII autophosphorylation.

The alpha-isoform of calcium/calmodulin-dependent kinase II (αCaMKII) is a major synaptic kinase that undergoes autophosphorylation after NMDA receptor activation, switching the kinase into a calcium-independent activity state. This αCaMKII autophosphorylation is essential for NMDA receptor-dependent long-term potentiation (LTP), induced by a single tetanus, in hippocampal area CA1 and in neocortex. Furthermore, the αCaMKII autophosphorylation is essential for contextual long-term memory (LTM) formation after a single training trial but not after a massed training session. Here, we show that in the absence of αCaMKII autophosphorylation contextual fear conditioning is hippocampus dependent and that multi-tetanus-dependent late-LTP cannot be induced in hippocampal area CA1. Furthermore, we show that in the absence of αCaMKII autophosphorylation contextual LTM persists for 30 days, the latest time point tested. Additionally, contextual, but not cued, LTM formation in the absence of αCaMKII autophosphorylation appears to be impaired in 18 month-old mice. Taken together, our findings suggest that αCaMKII autophosphorylation-independent plasticity in the hippocampus is sufficient for contextual LTM formation and that αCaMKII autophosphorylation may be important for delaying age-related impairments in hippocampal memory formation. Furthermore, they propose that NMDA receptor-dependent LTP in hippocampal area CA1 is essential for contextual LTM formation after a single trial but not after massed training. Finally, our results challenge the proposal that NMDA receptor-dependent LTP in neocortex is required for remote contextual LTM.

Novel insights into CaMKII function and regulation during memory formation.

Ca(2+)/calmodulin-dependent kinase II (CaMKII) is an abundant synaptic signalling molecule that is essential for memory formation and the induction of synaptic potentiation. Additionally, CaMKII plays a prominent role in synaptic tagging and metaplasticity. These abilities are mediated by kinase activity as well as binding to a wide variety of synaptic proteins, including NMDA receptor subunits, modulating CaMKII location and activity. A characteristic feature is that autophosphorylation of CaMKII switches the kinase into autonomous activity. Since CaMKII can be autonomously active and because CaMKII is required for the formation of memory it is important that the kinase activity is adequately switched off. However, the exact time window of increased activity and how this is terminated, it is still matter of debate. After training in a memory task CaMKII activity is increased for at least 30 min. This CaMKII activity and further activation of CaMKII may be regulated by changes in the expression of two endogenous CaMKII inhibitor proteins, CaMKII inhibitor Alpha and Beta, as they are up-regulated early after training. These endogenous inhibitors specifically block CaMKII activity and they inhibit the association with NMDA receptor subunits. Thus, regulation of the expression of endogenous CaMKII inhibitors may constitute a novel negative feedback on CaMKII signalling during memory formation.

The estrogen receptor-alpha-induced microRNA signature regulates itself and its transcriptional response.

Following estrogenic activation, the estrogen receptor-alpha (ERalpha) directly regulates the transcription of target genes via DNA binding. MicroRNAs (miRNAs) modulated by ERalpha have the potential to fine tune these regulatory systems and also provide an alternate mechanism that could impact on estrogen-dependent developmental and pathological systems. Through a microarray approach, we identify the subset of microRNAs (miRNAs) modulated by ERalpha, which include upregulation of miRNAs derived from the processing of the paralogous primary transcripts (pri-) mir-17-92 and mir-106a-363. Characterization of the mir-17-92 locus confirms that the ERalpha target protein c-MYC binds its promoter in an estrogen-dependent manner. We observe that levels of pri-mir-17-92 increase earlier than the mature miRNAs derived from it, implicating precursor cleavage modulation after transcription. Pri-mir-17-92 is immediately cleaved by DROSHA to pre-miR-18a, indicating that its regulation occurs during the formation of the mature molecule from the precursor. The clinical implications of this novel regulatory system were confirmed by demonstrating that pre-miR-18a was significantly upregulated in ERalpha-positive compared to ERalpha-negative breast cancers. Mechanistically, miRNAs derived from these paralogous pri-miRNAs (miR-18a, miR-19b, and miR-20b) target and downregulate ERalpha, while a subset of pri-miRNA-derived miRNAs inhibit protein translation of the ERalpha transcriptional p160 coactivator, AIB1. Therefore, different subsets of miRNAs identified act as part of a negative autoregulatory feedback loop. We propose that ERalpha, c-MYC, and miRNA transcriptional programs invoke a sophisticated network of interactions able to provide the wide range of coordinated cellular responses to estrogen.

Differential gene regulation by Epstein-Barr virus type 1 and type 2 EBNA2.

A transfection assay with a lymphoblastoid cell line infected with Epstein-Barr virus was used to compare the abilities of type 1 and type 2 EBNA2 to sustain cell proliferation. The reduced proliferation in cells expressing type 2 EBNA2 correlated with loss of expression of some cell genes that are known to be targets of type 1 EBNA2. Microarray analysis of EBNA2 target genes identified a small number of genes that are more strongly induced by type 1 than by type 2 EBNA2, and one of these genes (CXCR7) was shown to be required for proliferation of lymphoblastoid cell lines. The Epstein-Barr virus LMP1 gene was also more strongly induced by type 1 EBNA2 than by type 2, but this effect was transient. Type 1 and type 2 EBNA2 were equally effective at arresting cell proliferation of Burkitt's lymphoma cell lines lacking Epstein-Barr virus and were also shown to cause apoptosis in these cells. The results indicate that differential gene regulation by Epstein-Barr virus type 1 and type 2 EBNA2 may be the basis for the much weaker B-cell transformation activity of type 2 Epstein-Barr virus strains compared to type 1 strains.

Cell target genes of Epstein-Barr virus transcription factor EBNA-2: induction of the p55alpha regulatory subunit of PI3-kinase and its role in survival of EREB2.5 cells.

Microarray analysis covering most of the annotated RNAs in the human genome identified a panel of genes induced by the Epstein-Barr virus (EBV) EBNA-2 transcription factor in the EREB2.5 human B-lymphoblastoid cell line without the need for any intermediate protein synthesis. Previous data indicating that PIK3R1 RNA (the alpha regulatory subunit of PI3-kinase) was induced were confirmed, but it is now shown that it is the p55alpha regulatory subunit that is induced. Several EBV-immortalized lymphoblastoid cell lines were shown to express p55alpha. Expression of PI3-kinase p85 regulatory and p110 catalytic subunits was not regulated by EBNA-2. Proliferation of EREB2.5 lymphoblastoid cells was inhibited by RNAi knock-down of p55alpha protein expression, loss of p55alpha being accompanied by an increase in apoptosis. p55alpha is thus a functional target of EBNA2 in EREB2.5 cells and the specific regulation of p55alpha by EBV will provide an opportunity to investigate the physiological function of p55alpha in this human cell line.

Otx genes in the evolution of the vertebrate brain.

Only until a decade ago, animal phylogeny was traditionally based on the assumption that evolution of bilaterians went from simple to complex through gradual steps in which the extant species would represent grades of intermediate complexity that reflect the organizational levels of their ancestors. The advent of more sophisticated molecular biology techniques combined to an increasing variety of functional experiments has provided new tools, which lead us to consider evolutionary studies under a brand new light. An ancestral versus derived low-complexity of a given organism has now to be carefully re-assessed and also the molecular data so far accumulated needs to be re-evaluated. Conserved gene families expressed in the nervous system of all the species have been extensively used to reconstruct evolutionary steps, which may lead to identify the morphological as well as molecular features of the last common ancestor of bilaterians (Urbilateria). The Otx gene family is among these and will be here reviewed.