Changes in state of adenylation and time course of degradation of maternal mRNAs during oocyte maturation and early embryonic development in the mouse

Previous work has shown that more than 50% or about 50 pg of polyadenylated RNA found in the full-grown mouse oocyte is deadenylated or degraded during meiotic maturation. Here we show that rRNA declines by 60 pg during this period, accounting for most of the 80-pg decline in total RNA and indicating that a significant amount of mRNA is deadenylated but not degraded during maturation. Actin mRNA is deadenylated at about 7 hr of in vitro maturation, following the decline in its translation. The poly(A) tail on hypoxanthine phosphoribosyltransferase (HPRT) mRNA is elongated at 7 hr of maturation, preceding an increase in HPRT activity. Actin mRNA is partially degraded in the one-cell embryo and falls to near the limit of detection in the late two-cell stage, while HPRT mRNA shows no change in early two-cell embryos, but is deadenylated and declines greatly during the two-cell stage. In aging unfertilized eggs, most of these changes occur on a delayed schedule. The various species of alpha-tubulin mRNA are largely deadenylated and more than half are degraded during maturation. Taken together with other published results, we conclude that each mRNA has its own pattern of changes in the length of the poly(A) tail (correlated with translation) and degradation during the period of maternal control of protein synthesis, and, for those examined, the maternal mRNAs remaining in the early two-cell embryo are degraded to low levels by the late two-cell stage.     

Temperature-sensitive vaccinia virus mutants identify a gene with an essential role in viral replication.

Vaccinia virus mutants ts2 and ts25, members of the same complementation group, exhibit a temperature-dependent arrest at the stage of viral DNA replication. The lesions responsible for the mutant phenotypes have been localized to the far left region of the HindIII B genomic fragment by marker rescue studies. Hybrid selection analyses established that the DNA fragments positive for rescue represented the first open reading frame of the HindIII B fragment and encoded a 30-kilodalton protein. The gene is expressed early after infection as a rightwardly transcribed 1-kilobase-pair mRNA whose coordinates were determined by S1 nuclease mapping. To further the phenotypic analysis of the mutants, the accumulation of viral DNA sequences during permissive and nonpermissive infections was quantitated. The extent of the DNA- phenotype was shown to vary in different cell types. In mouse L cells at either high or low multiplicity of infection, nonpermissive DNA synthesis was less than 5% of that seen in permissive infections. This severe defect was mirrored by correspondingly low viral yields. In infections of BSC40 monkey cells, however, the deficiencies in both DNA synthesis and virus production were far less severe. For one mutant (ts2), the temperature sensitivity in BSC40 cells varied inversely with the multiplicity of infection.     

Vaccinia virus B1 kinase: phenotypic analysis of temperature-sensitive mutants and enzymatic characterization of recombinant proteins.

The vaccinia virus B1 gene encodes a 34-kDa protein with homology to protein kinases. In L cells infected nonpermissively with mutants containing lesions in the B1 gene (ts2 and ts25), the infectious cycle arrests prior to DNA replication. In this report, we demonstrate that DNA synthesis ceases when cultures infected with these mutants at 32 degrees C are shifted to the nonpermissive temperature (39.5 degrees C) in the midst of DNA replication. We also show that B1 protein is synthesized transiently during the early phase of infection, even when the progression to later stages of gene expression is prevented. Although wild-type (wt) B1 is stable, the ts B1 proteins are markedly labile in both L and BSC40 cells at both permissive and nonpermissive temperatures. These results suggest that the ts phenotype of the mutants is complex and may in part reflect a temperature-dependent requirement for kinase activity, an induction of temperature sensitivity in B1 substrates under nonpermissive conditions, and/or ts complementation by host factors. To facilitate biochemical analyses, recombinant wt B1, ts2 B1, and ts25 B1 were produced in Escherichia coli. The wt protein was able to phosphorylate serine and threonine residues on several exogenous substrates in vitro. The activity of ts25 B1 was 3% that of the wt enzyme, and no detectable kinase activity was associated with ts2 B1. In light of the inactivity of the ts2 B1 protein in vitro and its extreme lability in vivo, we attempted to isolate a vaccinia virus B1 null mutant by targeted interruption of the B1 gene at 32 degrees C. No null mutants were isolated. These results indicate that the B1 protein kinase provides a vital function which cannot be supplied by the host or circumvented by incubation at 32 degrees C.     

Optimizing the production of transformed pea (Pisum sativum L.) callus using disarmed Agrobacterium tumefaciens strains

For optimization of the transformation procedure with Pisum sativum L. stern explant callus was used to test the effect of disarmed Agrobacterium tumefaciens strains, cocultivation procedures (preconditioning of explants; use of Nicotiana tabacum L. nurse cultures), duration of cocultivation (2, 3 or 4 days), and agents for selection (kanamycin or hygromycin). The succinamopine strain EHA101(pBI1042) produced the highest percentage of transformed calli (77%) when used in conjunction with tobacco nurse culture during four days of cocultivation. Using this strain, kanamycin (76%) and hygromycin (77%) were equally effective selective agents, but for strain LBA4404(pBI1042) percentage of transformed calli was higher for hygromycin (63%) than for kanamycin (17%). The procedures and strains shown to be optimal for transformation of pea callus will now be complemented by a pea regeneration system.     

Occupational illness and poison control centers. Referral patterns and service needs.

In a study of occupational illness reported to a regional poison control center and to gauge the center''s outreach and services, we did follow-up interviews of 301 case contacts over a 6-month period. We ascertained referral routes, reasons for contacting the poison control center, and awareness of the center''s function. For 122 cases a nonphysician was the initial poison control center contact. Of the nonphysician contacts, 41 had already consulted a health care provider and been referred to the poison control center for assistance. Of the 70 persons with exposure, only 21 had been aware before their exposures that poison control center services might include occupational chemical illness consultation. Physicians and nonphysicians expressed similar reasons for contacting the poison control center, with 118 of 301 identifying the need for an exposure hazard risk assessment. These data suggest that although those contacting a poison control center because of occupational illness include a variety of cases, they have many similar service needs.     

Modeling Cumulative Effects of Climate and Development on Moose,Wolf, and Caribou Populations

Wildlife models focused solely on a single strong influence (e.g., habitat components, wildlife harvest) are limited in their ability to detect key mechanisms influencing population change. Instead, we propose integrated modeling in the context of cumulative effects assessment using multispecies population dynamics models linked to landscape-climate simulation at large spatial and temporal scales. We developed an integrated landscape and population simulation model using ALCES Online as the model-building platform, and the model accounted for key ecological components and relationships among moose (Alces alces), grey wolves (Canis lupus nubilus), and woodland caribou (Rangifer tarandus caribou) in northern Ontario, Canada. We simulated multiple scenarios over 5 decades (beginning 2020) to explore sensitivity to climate change and land use and assessed effects at multiple scales. The magnitude of effect and the relative importance of key factors (climate change, roads, and habitat) differed depending on the scale of assessment. Across the full extent of the study area (654,311km² [ecozonal scale]), the caribou population declined by 26% largely because of climate change and associated predator-prey response, which led to caribou range recession in the southern part of the study area. At the caribou range scale (108,378 km²), which focused on 2 herds in the northern part of the study area, climate change led to a 10% decline in the population and development led to an additional 7% decline. At the project scale (8,331 km²), which was focused more narrowly on the landscape surrounding 4 proposed mines, the caribou population declined by 29% largely in response to simulated development. Given that observed caribou population dynamics were sensitive to the cumulative effects of climate change, land use, interspecific interactions, and scale, insights from the analysis might not emerge under a less complex model. Our integrated modeling framework provides valuable support for broader regional assessments, including estimation of risk to caribou and Indigenous food security, and for developing and evaluating potential caribou recovery strategies. © 2021 The Authors. The Journal of Wildlife Management published by Wiley Periodicals LLC on behalf of The Wildlife Society.     

The Natural Product Magnolol as a Lead Structure for the Development of Potent Cannabinoid Receptor Agonists

Magnolol (4-allyl-2-(5-allyl-2-hydroxyphenyl)phenol), the main bioactive constituent of the medicinal plant Magnolia officinalis, and its main metabolite tetrahydromagnolol were recently found to activate cannabinoid (CB) receptors. We now investigated the structure-activity relationships of (tetrahydro)magnolol analogs with variations of the alkyl chains and the phenolic groups and could considerably improve potency. Among the most potent compounds were the dual CB₁/CB₂ full agonist 2-(2-methoxy-5-propyl-phenyl)-4-hexylphenol (61a, K _i CB₁∶0.00957 µM; K _i CB₂∶0.0238 µM), and the CB₂-selective partial agonist 2-(2-hydroxy-5-propylphenyl)-4-pentylphenol (60, K _i CB₁∶0.362 µM; K _i CB₂∶0.0371 µM), which showed high selectivity versus GPR18 and GPR55. Compound 61b, an isomer of 61a, was the most potent GPR55 antagonist with an IC₅₀ value of 3.25 µM but was non-selective. The relatively simple structures, which possess no stereocenters, are easily accessible in a four- to five-step synthetic procedure from common starting materials. The central reaction step is the well-elaborated Suzuki-Miyaura cross-coupling reaction, which is suitable for a combinatorial chemistry approach. The scaffold is versatile and may be fine-tuned to obtain a broad range of receptor affinities, selectivities and efficacies.     

Impact of seasonality,storage of semen,and sperm head‐shape on whole tissue methylation and expression of methylation responsive candidate genes in swine placenta and fetal livers from summer and winter breedings

Epigenetics includes the study of external factors that can influence the expression of genes by altering the accessibility of DNA through methylation. To investigate the epigenetic influence of season, sperm head shape, and semen storage on placental and fetal tissues, pregnancies were generated in the summer or winter using boar semen from either least or most sperm head shape change, collected during cool or warm seasons, and stored as cooled‐extended or cryopreserved. The lowest (p < 0.05) ratios of 5‐methylcytosine to 5‐hydroxymethylcytosine activity (5mC:5hmC) in fetal liver were from summer breedings and in placental tissues from winter breedings. The relative expression of placental CDH1 tended ( p < 0.10) to be greater in placenta generated from cryopreserved semen or semen collected during cool periods. The relative expression of placental GNAS was affected ( p < 0.05) by the interaction of breeding and semen collection seasons. Cryopreserved semen increased ( p < 0.05) the placental relative expression of GNAS. Placental MEST and RHOBTB3 tended ( p < 0.10) to have a greater relative expression from pregnancies generated using semen collected during cool periods used during winter breedings. Within fetal liver, the relative expression of GNAS and HGF was greater ( p < 0.05) from winter breedings. Interaction of winter breedings and least sperm head shape change tended ( p < 0.10) to have the greatest fetal liver expression of CDH1. Seasonality of semen collection, breeding, and the effect on sperm head shape change had an influence on the expression of genes with known differentially methylated regions or response to methylation activity from embryonic and extraembryonic tissues.