Select Small Core Structure Carbamates Exhibit High Contact Toxicity to ��Carbamate-Resistant�� Strain Malaria Mosquitoes, Anopheles gambiae (Akron)

Acetylcholinesterase (AChE) is a proven target for control of the malaria mosquito (Anopheles gambiae). Unfortunately, a single amino acid mutation (G119S) in An. gambiae AChE-1 (AgAChE) confers resistance to the AChE inhibitors currently approved by the World Health Organization for indoor residual spraying. In this report, we describe several carbamate inhibitors that potently inhibit G119S AgAChE and that are contact-toxic to carbamate-resistant An. gambiae. PCR-RFLP analysis was used to confirm that carbamate-susceptible G3 and carbamate-resistant Akron strains of An. gambiae carry wild-type (WT) and G119S AChE, respectively. G119S AgAChE was expressed and purified for the first time, and was shown to have only 3% of the turnover number (k _cat) of the WT enzyme. Twelve carbamates were then assayed for inhibition of these enzymes. High resistance ratios (>2,500-fold) were observed for carbamates bearing a benzene ring core, consistent with the carbamate-resistant phenotype of the G119S enzyme. Interestingly, resistance ratios for two oxime methylcarbamates, and for five pyrazol-4-yl methylcarbamates were found to be much lower (4- to 65-fold). The toxicities of these carbamates to live G3 and Akron strain An. gambiae were determined. As expected from the enzyme resistance ratios, carbamates bearing a benzene ring core showed low toxicity to Akron strain An. gambiae (LC₅₀>5,000 μg/mL). However, one oxime methylcarbamate (aldicarb) and five pyrazol-4-yl methylcarbamates (4a–e) showed good to excellent toxicity to the Akron strain (LC₅₀ = 32–650 μg/mL). These results suggest that appropriately functionalized “small-core” carbamates could function as a resistance-breaking anticholinesterase insecticides against the malaria mosquito.     

Gene repeat expansion and contraction by spontaneous intrachromosomal homologous recombination in mammalian cells

Homologous recombination (HR) is important in repairing errors of replication and other forms of DNA damage. In mammalian cells, potential templates include the homologous chromosome, and after DNA replication, the sister chromatid. Previous work has shown that the mammalian recombination machinery is organized to suppress interchromosomal recombination while preserving intrachromosomal HR. In the present study, we investigated spontaneous intrachromosomal HR in mouse hybridoma cell lines in which variously numbered tandem repeats of the µ heavy chain constant (Cµ) region reside at the haploid, chromosomal immunoglobulin µ heavy chain locus. This organization provides the opportunity to investigate recombination between homologous gene repeats in a well-defined chromosomal locus under conditions in which recombinants are conveniently recovered. This system revealed several features about the mammalian intrachromosomal HR process: (i) the frequency of HR was high (recombinants represented as much as several percent of the total of recombinants and non-recombinants); (ii) the recombination process appeared to be predominantly non-reciprocal, consistent with the possibility of gene conversion; (iii) putative gene conversion tracts were long (up to 13.4 kb); (iv) the recombination process occurred with precision, initiating and terminating within regions of shared homology. The results are discussed with respect to mammalian intrachromosomal HR involving interactions both within and between sister chromatids.     

Dendritic spine geometry: functional implication and regulation

Dendritic spines are tiny protrusions on dendritic shafts where most excitatory synapses are located. Recent advances in imaging technologies have given us great insight into the function of spines as biochemical compartments. Here we review recent evidence suggesting that the geometry of dendritic spines controls postsynaptic calcium signaling and is bidirectionally regulated during synaptic plasticity.     

Crowding does not affect monarch butterflies’ resistance to a protozoan parasite

Host density is an important factor when it comes to parasite transmission and host resistance. Increased host density can increase contact rate between individuals and thus parasite transmission. Host density can also cause physiological changes in the host, which can affect host resistance. Yet, the direction in which host density affects host resistance remains unresolved. It is also unclear whether food limitation plays a role in this effect. We investigated the effect of larval density in monarch butterflies, Danaus plexippus, on the resistance to their natural protozoan parasite Ophryocystis elektroscirrha under both unlimited and limited food conditions. We exposed monarchs to various density treatments as larvae to mimic high densities observed in sedentary populations. Data on infection and parasite spore load were collected as well as development time, survival, wing size, and melanization. Disease susceptibility under either food condition or across density treatments was similar. However, we found high larval density impacted development time, adult survival, and wing morphology when food was limited. This study aids our understanding of the dynamics of environmental parasite transmission in monarch populations, which can help explain the increased prevalence of parasites in sedentary monarch populations compared to migratory populations.     

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.Download video file.^{(51M, mov)}     

Translationally repressed mRNA transiently cycles through stress granules during stress

In mammals, repression of translation during stress is associated with the assembly of stress granules in the cytoplasm, which contain a fraction of arrested mRNA and have been proposed to play a role in their storage. Because physical contacts are seen with GW bodies, which contain the mRNA degradation machinery, stress granules could also target arrested mRNA to degradation. Here we show that contacts between stress granules and GW bodies appear during stress-granule assembly and not after a movement of the two preassembled structures. Despite this close proximity, the GW body proteins, which in some conditions relocalize in stress granules, come from cytosol rather than from adjacent GW bodies. It was previously reported that several proteins actively traffic in and out of stress granules. Here we investigated the behavior of mRNAs. Their residence time in stress granules is brief, on the order of a minute, although stress granules persist over a few hours after stress relief. This short transit reflects rapid return to cytosol, rather than transfer to GW bodies for degradation. Accordingly, most arrested mRNAs are located outside stress granules. Overall, these kinetic data do not support a direct role of stress granules neither as storage site nor as intermediate location before degradation.     

Different immune response of mice immunized with conjugates containing multiple copies of either consensus or mixotope versions of the V3 loop peptide from human immunodeficiency virus type 1

The critical role that antibody responses to the V3 loop epitope play in human immunodeficiency virus type 1 (HIV-1) neutralization has caused this peptide to be used in many HIV-1 vaccine candidates. To enhance cross-reactivity toward several V3 sequences, a database of 50 peptides of the V3 region from HIV-1 subtype A was used to design both a consensus peptide and a combinatorial peptide (mixotope) library representative of these sequences. The two immunogens (consensus and mixotope) were incorporated into multiple antigen peptide (MAP) constructions, conjugated to a recombinant surface antigen from hepatitis B virus (HbsAg) carrier protein, and inoculated to mice in combination with a C4 (CD4-binding) peptide MAP construction, also conjugated to HBsAg. The respective responses and cross-reactivity to several V3 loop sequences of both types of immunogens were compared. Mice inoculated with the V3 consensus-MAP-HBsAg + C4-MAP-HBsAg mixture elicited higher antibody responses than those given the V3 mixotope-MAP-HBsAg + C4-MAP-HBsAg mixture. In addition, pooled serum from the first group of immunogens analyzed at dilution 1:100 had higher cross-reactivity against V3 peptides on cellulose membranes than those from mice given the combinatorial immunogen. Fine epitope mapping of both consensus and C4 peptide by the spot synthesis technique showed that sera of the first group strongly recognized both sequences in their entirety, whereas mice immunized with the mixotope library recognized only the N-terminal region of V3. These results seem to suggest that the V3 consensus peptide is superior to the combinatorial strategy in inducing potent and cross-reactive responses to HIV.     

Identification of a new fatty acid synthesis-transport machinery at the peroxisomal membrane

The neurodegenerative disease X-linked adrenoleukodystrophy (X-ALD) is characterized by the abnormal accumulation of very long chain fatty acids. Mutations in the gene encoding the peroxisomal ATP-binding cassette half-transporter, adrenoleukodystrophy protein (ALDP), are the primary cause of X-ALD. To gain a better understanding of ALDP dysfunction, we searched for interaction partners of ALDP and identified binary interactions to proteins with functions in fatty acid synthesis (ACLY, FASN, and ACC) and activation (FATP4), constituting a thus far unknown fatty acid synthesis-transport machinery at the cytoplasmic side of the peroxisomal membrane. This machinery adds to the knowledge of the complex mechanisms of peroxisomal fatty acid metabolism at a molecular level and elucidates potential epigenetic mechanisms as regulatory processes in the pathogenesis and thus the clinical course of X-ALD.     

The Domain-Specific and Temperature-Dependent Protein Misfolding Phenotype of Variant Medium-Chain acyl-CoA Dehydrogenase

The implementation of expanded newborn screening programs reduced mortality and morbidity in medium-chain acyl-CoA dehydrogenase deficiency (MCADD) caused by mutations in the ACADM gene. However, the disease is still potentially fatal. Missense induced MCADD is a protein misfolding disease with a molecular loss-of-function phenotype. Here we established a comprehensive experimental setup to analyze the structural consequences of eight ACADM missense mutations (p.Ala52Val, p.Tyr67His, p.Tyr158His, p.Arg206Cys, p.Asp266Gly, p.Lys329Glu, p.Arg334Lys, p.Arg413Ser) identified after newborn screening and linked the corresponding protein misfolding phenotype to the site of side-chain replacement with respect to the domain. With fever being the crucial risk factor for metabolic decompensation of patients with MCADD, special emphasis was put on the analysis of structural and functional derangements related to thermal stress. Based on protein conformation, thermal stability and kinetic stability, the molecular phenotype in MCADD depends on the structural region that is affected by missense-induced conformational changes with the central β-domain being particularly prone to structural derangement and destabilization. Since systematic classification of conformational derangements induced by ACADM mutations may be a helpful tool in assessing the clinical risk of patients, we scored the misfolding phenotype of the variants in comparison to p.Lys329Glu (K304E), the classical severe mutation, and p.Tyr67His (Y42H), discussed to be mild. Experiments assessing the impact of thermal stress revealed that mutations in the ACADM gene lower the temperature threshold at which MCAD loss-of-function occurs. Consequently, increased temperature as it occurs during intercurrent infections, significantly increases the risk of further conformational derangement and loss of function of the MCAD enzyme explaining the life-threatening clinical courses observed during fever episodes. Early and aggressive antipyretic treatment thus may be life-saving in patients suffering from MCADD.