Imprinting: focusing on the center

Recent studies have focused on the identification of imprinting centers and on the elucidation of the mechanisms by which they control imprinting. These studies begin to shed light on the means by which imprinting marks are established in the gametes and on the various molecular strategies utilized to execute differential expression of the two parental alleles.     

Dimerization with Cannabinoid Receptors Allosterically Modulates Delta Opioid Receptor Activity during Neuropathic Pain

The diversity of receptor signaling is increased by receptor heteromerization leading to dynamic regulation of receptor function. While a number of studies have demonstrated that family A G-protein-coupled receptors are capable of forming heteromers in vitro, the role of these heteromers in normal physiology and disease has been poorly explored. In this study, direct interactions between CB₁ cannabinoid and delta opioid receptors in the brain were examined. Additionally, regulation of heteromer levels and signaling in a rodent model of neuropathic pain was explored. First we examined changes in the expression, function and interaction of these receptors in the cerebral cortex of rats with a peripheral nerve lesion that resulted in neuropathic pain. We found that, following the peripheral nerve lesion, the expression of both cannabinoid type 1 receptor (CB₁R) and the delta opioid receptor (DOR) are increased in select brain regions. Concomitantly, an increase in CB₁R activity and decrease in DOR activity was observed. We hypothesize that this decrease in DOR activity could be due to heteromeric interactions between these two receptors. Using a CB₁R-DOR heteromer-specific antibody, we found increased levels of CB₁R-DOR heteromer protein in the cortex of neuropathic animals. We subsequently examined the functionality of these heteromers by testing whether low, non-signaling doses of CB₁R ligands influenced DOR signaling in the cortex. We found that, in cortical membranes from animals that experienced neuropathic pain, non-signaling doses of CB₁R ligands significantly enhanced DOR activity. Moreover, this activity is selectively blocked by a heteromer-specific antibody. Together, these results demonstrate an important role for CB₁R-DOR heteromers in altered cortical function of DOR during neuropathic pain. Moreover, they suggest the possibility that a novel heteromer-directed therapeutic strategy for enhancing DOR activity, could potentially be employed to reduce anxiety associated with chronic pain.     

Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library

The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5′ and/or 3′ end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V_H families/segments was observed showing that ONCL did not introduce cloning biases for or against any V_H family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 × 10¹⁰ and by selecting five unique Fabs against GAPDH antigen.     

Public response to an anthrax attack: reactions to mass prophylaxis in a scenario involving inhalation anthrax from an unidentified source

An attack with Bacillus anthracis ("anthrax") is a known threat to the United States. When weaponized, it can cause inhalation anthrax, the deadliest form of the disease. Due to the rapid course of inhalation anthrax, delays in initiation of antibiotics may decrease survival chances. Because a rapid response would require cooperation from the public, there is a need to understand the public's response to possible mass dispensing programs. To examine the public's response to a mass prophylaxis program, this study used a nationally representative poll of 1,092 adults, supplemented by a targeted focus on 3 metropolitan areas where anthrax attacks occurred in 2001: New York City (n=517), Washington, DC (n=509), and Trenton/Mercer County, NJ (n=507). The poll was built around a "worst-case scenario" in which cases of inhalation anthrax are discovered without an identified source and the entire population of a city or town is asked to receive antibiotic prophylaxis within a 48-hour period. Findings from this poll provide important signs of public willingness to comply with public health recommendations for obtaining antibiotics from a dispensing site, although they also indicate that public health officials may face several challenges to compliance, including misinformation about the contagiousness of inhalation anthrax; fears about personal safety in crowds; distrust of government agencies to provide sufficient, safe, and effective medicine; and hesitation about ingesting antibiotic pills after receiving them. In general, people living in areas where anthrax attacks occurred in 2001 had responses similar to those of the nation as a whole.     

Receptor heteromerization expands the repertoire of cannabinoid signaling in rodent neurons

A fundamental question in G protein coupled receptor biology is how a single ligand acting at a specific receptor is able to induce a range of signaling that results in a variety of physiological responses. We focused on Type 1 cannabinoid receptor (CB₁R) as a model GPCR involved in a variety of processes spanning from analgesia and euphoria to neuronal development, survival and differentiation. We examined receptor dimerization as a possible mechanism underlying expanded signaling responses by a single ligand and focused on interactions between CB₁R and delta opioid receptor (DOR). Using co-immunoprecipitation assays as well as analysis of changes in receptor subcellular localization upon co-expression, we show that CB₁R and DOR form receptor heteromers. We find that heteromerization affects receptor signaling since the potency of the CB₁R ligand to stimulate G-protein activity is increased in the absence of DOR, suggesting that the decrease in CB₁R activity in the presence of DOR could, at least in part, be due to heteromerization. We also find that the decrease in activity is associated with enhanced PLC-dependent recruitment of arrestin3 to the CB₁R-DOR complex, suggesting that interaction with DOR enhances arrestin-mediated CB₁R desensitization. Additionally, presence of DOR facilitates signaling via a new CB₁R-mediated anti-apoptotic pathway leading to enhanced neuronal survival. Taken together, these results support a role for CB₁R-DOR heteromerization in diversification of endocannabinoid signaling and highlight the importance of heteromer-directed signal trafficking in enhancing the repertoire of GPCR signaling.     

Effect of RNA synthesis inhibitors on stimulation of sulfation by L-3,5,3'-triiodothyronine

Stimulation of sulfation by T₃¹ in chick embryo cartilage was blocked when actinomycin D or camptothecin was added to the incubation medium together with the T₃. When either inhibitor was added 2 hr after the T₃, the stimulation was not affected. The results suggest that there is an early step in the action of T₃ that involves synthesis of a stable species of RNA that is larger than 5S. α-Amanitin had no effect on the stimulation of sulfation by T₃, although it inhibited synthesis of poly(A)-rich RNA almost completely. This suggests that the action of T₃ is not associated with increased synthesis of RNA by RNA polymerase II. We conclude that the stimulation of sulfation by T₃ involves synthesis of a species of RNA that is larger than 5S but is not a messenger.     

Detection and characterization of cyanobacterial nifH genes.

The DNA sequence of a 359-bp fragment of nifH was determined for the heterocystous strains Anabaena sp. strain CA (ATCC 33047), Nostoc muscorum UTEX 1933, a Nostoc sp., Gloeothece sp. strain ATCC 27152, Lyngbya lagerheimii UTEX 1930, and Plectonema boryanum IU 594. Results confirmed that the DNA sequence of the 359-bp segment is sufficiently variable to distinguish cyanobacterial nifH genes from other eubacterial and arachaeobacterial nifH genes, as well as to distinguish heterocystous from nonheterocystous nifH genes. Nonheterocystous cyanobacterial nifH sequences were greater than 70 and 82% identical on the DNA and amino acid levels, respectively, whereas corresponding values for heterocystous cyanobacterial nifH sequences were 84 and 91%. The amplified nifH fragments can be used as DNA probes to differentiate between species, although there was substantial cross-reactivity between the nifH amplification products of some strains. However, an oligonucleotide designed from a sequence conserved within the heterocystous cyanobacteria hybridized primarily with the amplification product from heterocystous strains. The use of oligonucleotides designed from amplified nifH sequences shows great promise for characterizing assemblages of diazotrophs.     

Stress relaxation of porcine gluteus muscle subjected to sudden transverse deformation as related to pressure sore modeling

Computational studies of deep pressure sores (DPS) in skeletal muscles require information on viscoelastic constitutive behavior of muscles, particularly when muscles are loaded transversally as during bone-muscle interaction in sitting and lying immobilized patients. In this study, we measured transient shear moduli G(t) of fresh porcine muscles in vitro using the indentation method. We employed a custom-made pneumatic device that allowed rapid (2000 mms) 4 mm indentations. We tested 8 gluteus muscles, harvested from 5 adult pigs. Each muscle was indented transversally (perpendicularly to the direction of fibers) at 3 different sites, 7 times per site, to obtain nonpreconditioned (NPC) and preconditioned (PC) G(t) data. Short-term (GS) and long-term (GL) shear moduli were obtained directly from experiments. We further fitted measured G(t) data to a biexponential equation G(t) = G1 x exp(-t/tau1)+ G2 x exp(-t/tau2) + Ginfinity, which provided good fit, visually and in terms of the correlation coefficients. Typically, plateau of the stress relaxation curves (defined as 10% difference from final GL) was evident approximately 20 s after indentation. Short-term shear moduli GS (mean NPC: 8509 Pa, PC: 5711 Pa) were greater than long-term moduli GL (NPC: 609 Pa, PC: 807 Pa) by about an order of magnitude. Statistical analysis of parameters showed that only G2 was affected by preconditioning, while GL, GS, Ginfinity, tau1, tau2, and G1 properties were unaffected. Since DPS develop over time scales of minutes to hours, but most stress relaxation occurs within approximately 20 s, the most relevant property for computational modeling is GL (mean approximately 700 Pa), which is, conveniently, unaffected by preconditioning.     

Systems approach to explore components and interactions in the presynapse

The application of proteomic techniques to neuroscientific research provides an opportunity for a greater understanding of nervous system structure and function. As increasing amounts of neuroproteomic data become available, it is necessary to formulate methods to integrate these data in a meaningful way to obtain a more comprehensive picture of neuronal subcompartments. Furthermore, computational methods can be used to make biologically relevant predictions from large proteomic data sets. Here, we applied an integrated proteomics and systems biology approach to characterize the presynaptic (PRE) nerve terminal. For this, we carried out proteomic analyses of presynaptically enriched fractions, and generated a PRE literature‐based protein–protein interaction network. We combined these with other proteomic analyses to generate a core list of 117 PRE proteins, and used graph theory‐inspired algorithms to predict 92 additional components and a PRE complex containing 17 proteins. Some of these predictions were validated experimentally, indicating that the computational analyses can identify novel proteins and complexes in a subcellular compartment. We conclude that the combination of techniques (proteomics, data integration, and computational analyses) used in this study are useful in obtaining a comprehensive understanding of functional components, especially low‐abundance entities and/or interactions in the PRE nerve terminal.