Can animals recall the past and plan for the future?

According to the 'mental time travel hypothesis' animals, unlike humans, cannot mentally travel backwards in time to recollect specific past events (episodic memory) or forwards to anticipate future needs (future planning). Until recently, there was little evidence in animals for either ability. Experiments on memory in food-caching birds, however, question this assumption by showing that western scrub-jays form integrated, flexible, trial-unique memories of what they hid, where and when. Moreover, these birds can adjust their caching behaviour in anticipation of future needs. We suggest that some animals have elements of both episodic-like memory and future planning.     

Rapid de-localization of actin leading edge components with BDM treatment

Background

2,3-Butanedione monoxime (BDM) has been widely used as a non-muscle myosin inhibitor to investigate the role of non-muscle myosinII in the process of actin retrograde flow and other actin cytoskeletal processes. Recent reports show that BDM does not inhibit any non-muscle myosins so far tested, including nm-myosinII, prompting the question, how were these process affected in BDM studies?

Results

We have found that treatment of mammalian cells with BDM for only 1 min blocks actin incorporation at the leading edge in a permeabilized cell system. We show that inhibition of actin incorporation occurs through de-localization of leading edge proteins involved in actin polymerization – the Arp2/3 complex, WAVE, and VASP – that de-localize concomitantly with the leading edge actin network.

Conclusion

De-localization of actin leading edge components by BDM treatment is a newly described effect of this compound. It may explain many of the results previously ascribed to inhibition of non-muscle myosinII by BDM, particularly in studies of leading edge dynamics. Though this effect of BDM is intriguing, future studies probing actin dynamics at the leading edge should use more potent and specific inhibitors.

     

Mactinin: a modulator of the monocyte response to inflammation

During inflammatory processes, monocytes leave the blood stream at increased rates and enter inflammation tissue, where they undergo phenotypic transformation to mature macrophages with enhanced phagocytic activity. alpha-Actinin, a cytoskeletal protein, is present in focal adhesion complexes and left in the microenvironment as a result of cell movement. Mactinin, a 31 kDa amino-terminal fragment of alpha-actinin, is generated by the degradation of extracellular alpha-actinin by monocyte-secreted urokinase. We have previously demonstrated that mactinin promotes monocyte/macrophage maturation. We now report that 0.5-10 nM mactinin has significant chemotactic activity for monocytes. Mactinin seems to be present in inflammatory arthritis synovial fluid, because affinity-purified antisera reacted with a protein of the expected molecular mass in various types of arthritis fluids that were immunoaffinity-purified and subjected to Western analysis. Thus, six of seven samples from patients with psoriatic arthritis, reactive arthritis, gout, or ankylosing spondylitis contained mactinin at levels that are active in vitro. Initially, mactinin was not found in affinity-purified rheumatoid arthritis samples. However, it was detectable after the dissociation of immune complexes, suggesting that it was complexed to anti-microfilament auto-antibodies. In addition, mactinin was found in the lavage fluid from the arthritic knee joints of rabbits with antigen-induced arthritis and was absent from the contralateral control knee fluids. We conclude that mactinin is present in several types of inflammatory arthritis and might modulate mononuclear phagocyte response to inflammation.     

Directly observed therapy (DOT) for individuals with HIV: successes and challenges

Many HIV-infected individuals have not reaped the benefits of combination antiretroviral therapy due to inability either to adhere to medications or to access care. It is now recognized that innovative approaches are needed to increase access and adherence to highly active antiretroviral therapy (HAART), especially among these hard-to-reach populations. Due to the success of directly observed therapy (DOT) for the treatment of Mycobacterium tuberculosis (TB), our group and others have questioned whether DOT can be adapted to deliver HAART to hard-to-reach communities. In this review, we discuss the results of pilot programs that have utilized DOT in multiple different settings and use case studies to explore the diverse issues that can arise when implementing these programs. As we continue to gain more experience with observed therapy, we will be able to better identify the key components for a successful intervention.     

Progress towards single-molecule DNA sequencing: a one color demonstration

Single molecules of fluorescently labeled nucleotides were detected during the cleavage of individual DNA fragments by a processive exonuclease. In these experiments, multiple (10-100) strands of DNA with tetramethyl rhodamine labeled dUMP (TMR-dUMP) incorporated into the sequence were anchored in flow upstream of the detection region of an ultra sensitive flow cytometer. A dilute solution of Exonuclease I passed over the microspheres. When an exonuclease attached to a strand, processive digestion of that strand began. The liberated, labeled bases flowed through the detection region and were detected at high efficiency at the single-molecule level by laser-induced fluorescence. The digestion of a single strand of DNA by a single exonuclease was discernable in these experiments. This result demonstrates the feasibility of single-molecule DNA sequencing. In addition, these experiments point to a new and practical means of arriving at a consensus sequence by individually reading out identical sequences on multiple fragments.     

Detection and differentiation of yellow head complex viruses using monoclonal antibodies

Three monoclonal antibodies (MAbs) raised against pathogenic yellow head virus (YHV) from Thailand were tested against tissues of shrimp from Thailand, Australia, Ecuador and India that were purported to be infected with yellow head complex viruses. MAbs V-3-2B and Y-18 were specific to gp116 and gp64 envelope proteins, respectively, while Y-19 was specific to a 20 kDa putative nucleoprotein p20. As a preliminary step, the site of reactivity of the 3 MAbs in YHV was determined by immuno-electron microscopy using ultra-thin sections of YHV-infected shrimp tissue and negatively stained, semi-purified YHV particles. As expected, MAb Y-19 reacted with viral nucleocapsids in ultra-thin sections but not with negatively stained, whole virions; MAb V-3-2B did react with negatively stained, whole virions, but not with virions or nucleocapsids in ultra-thin sections. Unexpectedly, MAb Y-18 did not react with whole or sectioned virions. By immunohistochemistry, MAbs Y-19 and Y-18 reacted with Penaeus monodon tissues infected with either YHV or with gill-associated virus (GAV) from Australia, while MAb V-3-2B reacted with YHV only. In addition, all the YHV and GAV tissue samples gave positive in situ hybridization reactions with a cDNA probe specific to the ORF1b gene of YHV. They also gave expected differential RT-PCR results for YHV and GAV. By contrast, 2 natural Thai shrimp specimens with no gross signs of disease gave similar immunohistochemical reactions and RT-PCR reactions to GAV. However, sequencing of their RT-PCR products showed that they shared 92.7% identity with GAV, but only 79.0% identity with YHV. Although specimens from Ecuador and India displayed histopathology suggestive of YHV infection, they gave negative immunohistochemical reactions with all 3 Mabs, and negative in situ hybridization results. Additional work is required to determine whether a virus from the yellow head complex was responsible for their observed histopathology. These data show that the 3 YHV MAbs could be used in diagnostic situations to differentiate some viruses in the yellow head virus complex.     

Selective toxicity of engineered lentivirus lytic peptides in a CF airway cell model

Lentivirus lytic peptides (LLPs) are derived from HIV-1 and have antibacterial properties. LLP derivatives (eLLPs) were engineered for greater potency against Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA). Minimum bactericidal concentration (MBC) was determined in low and physiologic salt concentrations. MBC was decreased against SA and equivalent against PA in physiologic salt when compared to the parent compound LLP1. In a novel cystic fibrosis (CF) airway cell model, one derivative, WLSA5, reduced the number of adherent PA and only moderately affected CF cell viability. Overall, eLLPs are selectively toxic to bacteria and may be useful against CF airway infections.     

Extracellular signal-regulated kinase 7, a regulator of hormone-dependent estrogen receptor destruction

Estrogen receptor alpha (ER alpha) degradation is regulated by ubiquitination, but the signaling pathways that modulate ER alpha turnover are unknown. We found that extracellular signal-regulated kinase 7 (ERK7) preferentially enhances the destruction of ER alpha but not the related androgen receptor. Loss of ERK7 was correlated with breast cancer progression, and all ER alpha-positive breast tumors had decreased ERK7 expression compared to that found in normal breast tissue. In human breast cells, a dominant-negative ERK7 mutant decreased the rate of endogenous ER alpha degradation >4-fold in the presence of hormone and potentiated estrogen responsiveness. ERK7 targets the ER alpha ligand-binding domain for destruction by enhancing its ubiquitination. Thus, ERK7 is a novel regulator of estrogen responsiveness through its control of ER alpha turnover.     

MEK kinase 2 and the adaptor protein Lad regulate extracellular signal-regulated kinase 5 activation by epidermal growth factor via Src

Lad is an SH2 domain-containing adaptor protein that binds MEK kinase 2 (MEKK2), a mitogen-activated protein kinase (MAPK) kinase kinase for the extracellular signal-regulated kinase 5 (ERK5) and JNK pathways. Lad and MEKK2 are in a complex in resting cells. Antisense knockdown of Lad expression and targeted gene disruption of MEKK2 expression results in loss of epidermal growth factor (EGF) and stress stimuli-induced activation of ERK5. Activation of MEKK2 and the ERK5 pathway by EGF and stress stimuli is dependent on Src kinase activity. The Lad-binding motif is encoded within amino acids 228 to 282 in the N terminus of MEKK2, and expression of this motif blocks Lad-MEKK2 interaction, resulting in inhibition of Src-dependent activation of MEKK2 and ERK5. JNK activation by EGF is similarly inhibited by loss of Lad or MEKK2 expression and by blocking the interaction of MEKK2 and Lad. Our studies demonstrate that Src kinase activity is required for ERK5 activation in response to EGF, MEKK2 expression is required for ERK5 activation by Src, Lad and MEKK2 association is required for Src activation of ERK5, and EGF and Src stimulation of ERK5-regulated MEF2-dependent promoter activity requires a functional Lad-MEKK2 signaling complex.     

Optical quantal analysis reveals a presynaptic component of LTP at hippocampal Schaffer-associational synapses

The mechanisms by which long-term potentiation (LTP) is expressed are controversial, with evidence for both presynaptic and postsynaptic involvement. We have used confocal microscopy and Ca(2+)-sensitive dyes to study LTP at individual visualized synapses. Synaptically evoked Ca(2+) transients were imaged in distal dendritic spines of pyramidal cells in cultured hippocampal slices, before and after the induction of LTP. At most synapses, from as early as 10 min to at least 60 min after induction, LTP was associated with an increase in the probability of a single stimulus evoking a postsynaptic Ca(2+) response. These observations provide compelling evidence of a presynaptic component to the expression of early LTP at Schaffer-associational synapses. In most cases, the store-dependent evoked Ca(2+) transient in the spine was also increased after induction, a novel postsynaptic aspect of LTP.