Use of formylated yeast initiator Met tRNA to define the NH2-terminal residues of rat preproinsulin and pregrowth hormone

A method for unambiguously determining the initiator methionine residue and the adjacent NH2-terminal amino acid sequence of cell-free translation products of eukaryotic messenger RNA is described. In this procedure, the NH2 termini of nascent peptides are blocked by incorporating labeled formylmethionine instead of methionine, using yeast initiator tRNA in the wheat germ cell-free system. After immunoprecipitation of the desired product the radiolabeled material is treated with dansyl-Cl to irreversibly block all remaining free amino groups. The material is then deformylated by mild acid hydrolysis and subjected to automated Edman degradation. Only those products that had been synthesized with formylmethionine residues at their NH2-termini can then give rise to labeled phenylthiohydantoin derivatives during degradation. Using this method, we have defined the initiation sites in both rat preproinsulin and pregrowth hormone messenger RNAs.     

N-formyl-L-methionine deformylase activity in human leucocytes and platelets.

Extracts of human neutrophils, lymphocytes and platelets enzymically deformylate N-formyl-L-methionine. Enzyme activity is stimulated by Co2+, inhibited by bivalent-cation chelators and unaffected by inhibitors of serine, thiol and carboxyl proteinases. Leucocyte or platelet N-formylmethionine deformylase may be important in modulation of neutrophil responses to chemoattractant formylmethionyl peptides or similar compounds.     

Determination of base pairing in Escherichia coli and Bacillus stearothermophilus 5S RNAs by infrared spectroscopy.

The extent of base pairing in Escherichia coli and Bacillus stearothermophilus 5S RNAs was determined by infrared spectroscopy. From the infrared spectra taken at 20 degrees and 52 degrees C it is concluded that E. coli and B. stearothermophlius 5S RNAs possess a large number of base pairs (Table I). Comparison of our results with those previously published using other methods leads to the conclusion that the structures of prokaryotic 5S RNAs involve a large number of tertiary interactions, in which the base pairing is not necessarily solely of the Watson-Crick type.     

An Excess of Deleterious Variants in VEGF-A Pathway Genes in Down-Syndrome-Associated Atrioventricular Septal Defects

About half of people with trisomy 21 have a congenital heart defect (CHD), whereas the remainder have a structurally normal heart, demonstrating that trisomy 21 is a significant risk factor but is not causal for abnormal heart development. Atrioventricular septal defects (AVSD) are the most commonly occurring heart defects in Down syndrome (DS), and ∼65% of all AVSD is associated with DS. We used a candidate-gene approach among individuals with DS and complete AVSD (cases = 141) and DS with no CHD (controls = 141) to determine whether rare genetic variants in genes involved in atrioventricular valvuloseptal morphogenesis contribute to AVSD in this sensitized population. We found a significant excess (p < 0.0001) of variants predicted to be deleterious in cases compared to controls. At the most stringent level of filtering, we found potentially damaging variants in nearly 20% of cases but fewer than 3% of controls. The variants with the highest probability of being damaging in cases only were found in six genes: COL6A1, COL6A2, CRELD1, FBLN2, FRZB, and GATA5. Several of the case-specific variants were recurrent in unrelated individuals, occurring in 10% of cases studied. No variants with an equal probability of being damaging were found in controls, demonstrating a highly specific association with AVSD. Of note, all of these genes are in the VEGF-A pathway, even though the candidate genes analyzed in this study represented numerous biochemical and developmental pathways, suggesting that rare variants in the VEGF-A pathway might contribute to the genetic underpinnings of AVSD in humans.     

A study on the minimum number of loci required for genetic evaluation using a finite locus model

For a finite locus model, Markov chain Monte Carlo (MCMC) methods can be used to estimate the conditional mean of genotypic values given phenotypes, which is also known as the best predictor (BP). When computationally feasible, this type of genetic prediction provides an elegant solution to the problem of genetic evaluation under non-additive inheritance, especially for crossbred data. Successful application of MCMC methods for genetic evaluation using finite locus models depends, among other factors, on the number of loci assumed in the model. The effect of the assumed number of loci on evaluations obtained by BP was investigated using data simulated with about 100 loci. For several small pedigrees, genetic evaluations obtained by best linear prediction (BLP) were compared to genetic evaluations obtained by BP. For BLP evaluation, used here as the standard of comparison, only the first and second moments of the joint distribution of the genotypic and phenotypic values must be known. These moments were calculated from the gene frequencies and genotypic effects used in the simulation model. BP evaluation requires the complete distribution to be known. For each model used for BP evaluation, the gene frequencies and genotypic effects, which completely specify the required distribution, were derived such that the genotypic mean, the additive variance, and the dominance variance were the same as in the simulation model. For lowly heritable traits, evaluations obtained by BP under models with up to three loci closely matched the evaluations obtained by BLP for both purebred and crossbred data. For highly heritable traits, models with up to six loci were needed to match the evaluations obtained by BLP.     

Timing,frequency, and duration of incubation recesses in dabbling ducks

Nest attendance is an important determinant of avian reproductive success, and identifying factors that influence the frequency and duration of incubation recesses furthers our understanding of how incubating birds balance their needs with those of their offspring. We characterized the frequency and timing (start time, end time, and duration) of incubation recesses for mallard (Anas platyrhynchos) and gadwall (Mareca strepera) hens breeding in Suisun Marsh, California, USA, and examined the influences of day of year, ambient temperature at the nest, incubation day, and clutch size on recess frequency and timing using linear mixed models. Mallard, on average, took more recesses per day (1.69 ± 0.80, mean ± standard deviation) than did gadwall (1.39 ± 0.69), and 45% of mallard nest‐days were characterized by two recesses, while only 27% of gadwall nest‐days were characterized by two recesses. Mallard morning recesses started at 06:14 ± 02:46 and lasted 106.11 ± 2.01 min, whereas mallard afternoon recesses started at 16:39 ± 02:11 and lasted 155.39 ± 1.99 min. Gadwall morning recesses started at 06:30 ± 02:46 and lasted 91.28 ± 2.32 min, and gadwall afternoon recesses started at 16:31 ± 01:57 and lasted 192.69 ± 1.89 min. Mallard and gadwall started recesses earlier in the day with increasing ambient temperature, but later in the day as the season progressed. Recess duration decreased as the season progressed and as clutch size increased, and increased with ambient temperature at the nest. The impending darkness of sunset appeared to be a strong cue for ending a recess and returning to the nest, because hens returned to their nests earlier than expected when recesses were expected to end after sunset. Within hens, the timing of incubation recesses was repeatable across incubation days and was most repeatable for mallard afternoon recesses and on days in which hens took only one recess. Hens were most likely to be away from nests between 04:00 and 07:00 and between 16:00 and 19:00; therefore, investigators should search for nests between 07:00 and 16:00. Our analyses identified important factors influencing incubation recess timing in dabbling ducks and have important implications for nest monitoring programs.     

Interrupted incubation: How dabbling ducks respond when flushed from the nest

Nesting birds must provide a thermal environment sufficient for egg development while also meeting self‐maintenance needs. Many birds, particularly those with uniparental incubation, achieve this balance through periodic incubation recesses, during which foraging and other self‐maintenance activities can occur. However, incubating birds may experience disturbances such as predator or human activity which interrupt natural incubation patterns by compelling them to leave the nest. We characterized incubating mallard Anas platyrhynchos and gadwall Mareca strepera hens’ responses when flushed by predators and investigators in Suisun Marsh, California, USA. Diurnal incubation recesses initiated by investigators approaching nests were 63% longer than natural diurnal incubation recesses initiated by the hen (geometric mean: 226.77 min versus 142.04 min). Nocturnal incubation recesses, many of which were likely the result of predators flushing hens, were of similar duration regardless of whether the nest was partially depredated during the event (115.33 [101.01;131.68] minutes) or not (119.62 [111.96;127.82] minutes), yet were 16% shorter than natural diurnal incubation recesses. Hens moved further from the nest during natural diurnal recesses or investigator‐initiated recesses than during nocturnal recesses, and the proportion of hen locations recorded in wetland versus upland habitat during recesses varied with recess type (model‐predicted means: natural diurnal recess 0.77; investigator‐initiated recess 0.82; nocturnal recess 0.31). Hens were more likely to take a natural recess following an investigator‐initiated recess earlier that same day than following a natural recess earlier that same day, and natural recesses that followed an investigator‐initiated recess were longer than natural recesses that followed an earlier natural recess, suggesting that hens may not fulfill all of their physiological needs during investigator‐initiated recesses. We found no evidence that the duration of investigator‐initiated recesses was influenced by repeated visits to the nest, whether by predators or by investigators, and trapping and handling the hen did not affect investigator‐initiated recess duration unless the hen was also fitted with a backpack‐harness style GPS–GSM transmitter at the time of capture. Hens that were captured and fitted with GPS–GSM transmitters took recesses that were 26% longer than recesses during which a hen was captured but a GPS–GSM transmitter was not attached. Incubation interruptions had measurable but limited and specific effects on hen behavior.     

A focus on fixation

Three fixation issues related to immunostaining are discussed here: 1) Generally, a tissue block is fixed, then embedded and sectioned (pre-fixation). The type of fixative applied, crosslinking or coagulating, has an impact on selecting an epitope retrieval method. Individual antigens have a fixation–retrieval characteristic. 2) A long fixation time, especially with crosslinking fixatives, may compromise the result of immunostaining. This negative effect varies among different antigens and can be partially restored by applying a more sensitive/efficient detection system such as tyramide amplification. 3) Sections cut from a fresh frozen tissue block usually are acetone fixed (post-fixation). This was accepted as the “gold standard” for a long time. Post-fixation, however, may have serious consequences for preservation of small peptides leaking from the cut open cells, whereas this is not the case with pre-fixed intact cells. Consequently, the concept of an acetone post-fixed cryostat tissue section as “gold standard” no longer exists and a more appropriate use of the terms immunohistochemistry and immunocytochemistry therefore seems justified. For many antibodies, it is not known whether a formalin fixed, paraffin embedded tissue specimen is appropriate. Suggestions are made for creating a positive control cell block for testing such antibodies.     

Corticotropin-releasing Factor Receptor 2 Mediates Sex-Specific Cellular Stress Responses

Although females suffer twice as much as males from stress-related disorders, sex-specific participating and pathogenic cellular stress mechanisms remain uncharacterized. Using corticotropin-releasing factor receptor 2–deficient (Crhr2^−/− ) and wild-type (WT) mice, we show that CRF receptor type 2 (CRF₂) and its high-affinity ligand, urocortin 1 (Ucn1), are key mediators of the endoplasmic reticulum (ER) stress response in a murine model of acute pancreatic inflammation. Ucn1 was expressed de novo in acinar cells of male, but not female WT mice during acute inflammation. Upon insult, acinar Ucn1 induction was markedly attenuated in male but not female Crhr2^−/− mice. Crhr₂^−/− mice of both sexes show exacerbated acinar cell inflammation and necrosis. Electron microscopy showed mild ER damage in WT male mice and markedly distorted ER structure in Crhr2^−/− male mice during pancreatitis. WT and Crhr2^−/− female mice showed similarly distorted ER ultrastructure that was less severe than distortion seen in Crhr2^−/− male mice. Damage in ER structure was accompanied by increased ubiquitination, peIF2, and mistargeted localization of vimentin in WT mice that was further exacerbated in Crhr2^−/− mice of both sexes during pancreatitis. Exogenous Ucn1 rescued many aspects of histological damage and cellular stress response, including restoration of ER structure in male WT and Crhr2^−/−mice, but not in females. Instead, females often showed increased damage. Thus, specific cellular pathways involved in coping and resolution seem to be distinct to each sex. Our results demonstrate the importance of identifying sex-specific pathogenic mechanisms and their value in designing effective therapeutics.