Molecular Cloning and Characterization of Juvenile Hormone Acid Methyltransferase in the Honey Bee,Apis mellifera,and Its Differential Expression during Caste Differentiation

Juvenile hormone acid methyltransferase (JHAMT) is an enzyme involved in one of the final steps of juvenile hormone biosynthesis in insects. It transfers a methyl group from S-adenosyl-L-methionine (SAM) to the carboxyl group of either farnesoic acid (FA) or JH acid (JHA). Several genes coding for JHAMT have been cloned and characterized from insects from different orders, and they have been shown to play critical roles in metamorphosis and reproduction. However, the significance of JHAMT in Hymenopteran insects is unknown. We used RACE amplification method to clone JHAMT cDNA from the honey bee, Apis mellifera (AmJHAMT). The full length cDNA of AmJHAMT that we cloned is 1253bp long and encodes a 278-aa protein that shares 32-36% identity with known JHAMTs. A SAM-binding motif, conserved in the SAM-dependent methyltransferase (SAM-MT) superfamily, is present in AmJHAMT. Its secondary structure also contains a typical SAM-MT fold. Most of the active sites bound with SAM and substrates (JHA or FA) are conserved in AmJHAMT as in other JHAMT orthologs. Phylogenetic analysis clustered AmJHAMT with the other orthologs from Hymenoptera to form a major clade in the phylogenetic tree. Purified recombinant AmJHAMT protein expressed in E. coli was used to produce polyclonal antibodies and to verify the identity of AmJHAMT by immunoblotting and mass spectrometry. Quantitative RT-PCR and immunoblotting analyses revealed that queen larvae contained significantly higher levels of AmJHAMT mRNA and protein than worker larvae during the periods of caste development. The temporal profiles of both AmJHAMT mRNA and protein in queens and workers showed a similar pattern as the JH biosynthesis. These results suggest that the gene that we cloned codes for a functional JHAMT that catalyzes the final reactions of JH biosynthesis in honey bees. In addition, AmJHAMT may play an important role in honey bee caste differentiation.     

Influenza Virus Reassortment Occurs with High Frequency in the Absence of Segment Mismatch

Reassortment is fundamental to the evolution of influenza viruses and plays a key role in the generation of epidemiologically significant strains. Previous studies indicate that reassortment is restricted by segment mismatch, arising from functional incompatibilities among components of two viruses. Additional factors that dictate the efficiency of reassortment remain poorly characterized. Thus, it is unclear what conditions are favorable for reassortment and therefore under what circumstances novel influenza A viruses might arise in nature. Herein, we describe a system for studying reassortment in the absence of segment mismatch and exploit this system to determine the baseline efficiency of reassortment and the effects of infection dose and timing. Silent mutations were introduced into A/Panama/2007/99 virus such that high-resolution melt analysis could be used to differentiate all eight segments of the wild-type and the silently mutated variant virus. The use of phenotypically identical parent viruses ensured that all progeny were equally fit, allowing reassortment to be measured without selection bias. Using this system, we found that reassortment occurred efficiently (88.4%) following high multiplicity infection, suggesting the process is not appreciably limited by intracellular compartmentalization. That co-infection is the major determinant of reassortment efficiency in the absence of segment mismatch was confirmed with the observation that the proportion of viruses with reassortant genotypes increased exponentially with the proportion of cells co-infected. The number of reassortants shed from co-infected guinea pigs was likewise dependent on dose. With 10⁶ PFU inocula, 46%–86% of viruses isolated from guinea pigs were reassortants. The introduction of a delay between infections also had a strong impact on reassortment and allowed definition of time windows during which super-infection led to reassortment in culture and in vivo. Overall, our results indicate that reassortment between two like influenza viruses is efficient but also strongly dependent on dose and timing of the infections.     

Human Pol ɛ-dependent replication errors and the influence of mismatch repair on their correction

Mutations in human DNA polymerase (Pol) ?, one of three eukaryotic Pols required for DNA replication, have recently been found associated with an ultramutator phenotype in tumors from somatic colorectal and endometrial cancers and in a familial colorectal cancer. Possibly, Pol ? mutations reduce the accuracy of DNA synthesis, thereby increasing the mutational burden and contributing to tumor development. To test this possibility in vivo, we characterized an active site mutant allele of human Pol ? that exhibits a strong mutator phenotype in vitro when the proofreading exonuclease activity of the enzyme is inactive. This mutant has a strong bias toward mispairs opposite template pyrimidine bases, particularly T•dTTP mispairs. Expression of mutant Pol ? in human cells lacking functional mismatch repair caused an increase in mutation rate primarily due to T•dTTP mispairs. Functional mismatch repair eliminated the increased mutagenesis. The results indicate that the mutant Pol ? causes replication errors in vivo, and is at least partially dominant over the endogenous, wild type Pol ?. Since tumors from familial and somatic colorectal patients arise with Pol ? mutations in a single allele, are microsatellite stable and have a large increase in base pair substitutions, our data are consistent with a Pol ? mutation requiring additional factors to promote tumor development.     

Role for the Epidermal Growth Factor Receptor in Chemotherapy-Induced Alopecia

Treatment of cancer patients with chemotherapeutics like cyclophosphamide often causes alopecia as a result of premature and aberrant catagen. Because the epidermal growth factor receptor (EGFR) signals anagen hair follicles to enter catagen, we hypothesized that EGFR signaling may be involved in cyclophosphamide-induced alopecia. To test this hypothesis, skin-targeted Egfr mutant mice were generated by crossing floxed Egfr and Keratin 14 promoter-driven Cre recombinase mice. Cyclophosphamide treatment of control mice resulted in alopecia while Egfr mutant skin was resistant to cyclophosphamide-induced alopecia. Egfr mutant skin entered catagen normally, as indicated by dermal papilla condensation and decreased follicular proliferation, but did not progress to telogen as did Egfr wild type follicles. Egfr mutant follicles responded with less proliferation, apoptosis, and fewer p53-positive cells after cyclophosphamide. Treatment of control mice with the EGFR inhibitors erlotinib or gefitinib similarly suppressed alopecia and catagen progression by cyclophosphamide. Secondary analysis of clinical trials utilizing EGFR-targeted therapies and alopecia-inducing chemotherapy also revealed evidence for involvement of EGFR in chemotherapy-induced alopecia. Taken together, our results demonstrated the involvement of EGFR signaling in chemotherapy-induced alopecia, which will help in the design of novel therapeutic regimens to minimize chemotherapy-induced alopecia.     

Transfected HEK293 Cells Expressing Functional Recombinant Intercellular Adhesion Molecule 1 (ICAM-1) – A Receptor Associated with Severe Plasmodium falciparum Malaria

Intercellular adhesion molecule 1 (ICAM-1) is a membrane-bound glycoprotein expressed on endothelial cells and cells of the immune system. Human ICAM-1 mediates adhesion and migration of leucocytes, and is implicated in inflammatory pathologies, autoimmune diseases and in many cancer processes. Additionally, ICAM-1 acts as receptor for pathogens like human rhinovirus and Plasmodium falciparum malaria parasites. A group of related P. falciparum erythrocyte membrane protein 1 (PfEMP1) domains, the DBLβ, mediates ICAM-1 binding of P. falciparum-infected erythrocytes. This ICAM‑1-binding phenotype has been suggested to be involved in the development of cerebral malaria. However, more studies identifying cross-reactive antibody and ICAM-1-binding epitopes and the establishment of a clinical link between DBLβ expression and e.g. cerebral malaria are needed before the DBLβ domains can be put forward as vaccine candidates and go into clinical trials. Such studies require availability of functional recombinant ICAM-1 in large quantities. In this study, we compared recombinant ICAM-1 expressed in HEK293 and COS-7 cells with mouse myeloma NS0 ICAM-1 purchased from a commercial vendor in terms of protein purity, yield, fold, ability to bind DBLβ, and relative cost. We present a HEK293 cell-based, high-yield expression and purification scheme for producing inexpensive, functional ICAM‑1. ICAM-1 expressed in HEK293 is applicable to malaria research and can also be useful in other research fields.     

MCHM Acts as a Hydrotrope,Altering the Balance of Metals in Yeast

Biological Trace Element Research - While drugs and other industrial chemicals are routinely studied to assess risks, many widely used chemicals have not been thoroughly evaluated. One such...     

Persistent anthropogenic legacies structure depth dependence of regenerating rooting systems and their functions

Biotically-mediated weathering helps to shape Earth’s surface. For example, plants expend carbon (C) to mobilize nutrients in forms whose relative abundances vary with depth. It thus is likely that trees’ nutrient acquisition strategies—their investment in rooting systems and exudates—may function differently following disturbance-induced changes in depth of rooting zones and soil nutrient stocks. These changes may persist across centuries. We test the hypothesis that plant C allocation for nutrient acquisition is depth dependent as a function of rooting system development and relative abundances of organic vs. mineral nutrient stocks. We further posit that patterns of belowground C allocation to nutrient acquisition reveal anthropogenic signatures through many decades of forest regeneration. To test this idea, we examined fine root abundances and rooting system C in organic acid exudates and exo-enzymes in tandem with depth distributions of organically- and mineral-bound P stocks. Our design permitted us to estimate C tradeoffs between organic vs. mineral nutrient benefits in paired forests with many similar aboveground traits but different ages: post-agricultural mixed-pine forests and older reference hardwoods. Fine roots were more abundant throughout the upper 2 m in reference forest soils than in regenerating stands. Rooting systems in all forests exhibited depth-dependent C allocations to nutrient acquisition reflecting relative abundances of organic vs. mineral bound P stocks. Further, organic vs. mineral stocks underwent redistribution with historic land use, producing distinct ecosystem nutritional economies. In reference forests, rooting systems are allocating C to relatively deep fine roots and low-C exudation strategies that can increase mobility of mineral-bound P stocks. Regenerating forests exhibit relatively shallower fine root distributions and more diverse exudation strategies reflecting more variable nutrient stocks. We observed these disparities in rooting systems’ depth and nutritional mechanisms even though the regenerating forests have attained aboveground biomass stocks similar to those in reference hardwood forests. These distinctions offer plausible belowground mechanisms for observations of continued C sink strength in relatively old forests, and have implications for soil C fates and soil development on timescales relevant to human lifetimes. As such, depth-dependent nutrient returns on plant C investments represent a subtle but consequential signal of the Anthropocene.