Diets enriched in unsaturated fatty acids enhance early embryonic development in lactating Holstein cows

We hypothesized that a diet enriched in alpha-linolenic acid would enhance embryonic development relative to diets enriched in linoleic or saturated fatty acids. Twenty-four lactating Holstein cows (86+/-22 d postpartum) were assigned to one of three diets containing saturated fatty acids (SAT; high in palmitic and stearic acids), whole flaxseed (FLX; high in alpha-linolenic acid) or sunflower seed (SUN; high in linoleic acid). Rations were formulated to provide 750 g supplemental fat/cow/d in all dietary groups. Ovulation (Day 0) was synchronized approximately 20 d after diets began. Ultrasound-guided follicular ablation of all follicles >8 mm was performed 5 d after ovulation; super stimulatory treatments began 2 d after follicular ablation, and embryos were collected non-surgically 7 d after AI. Fertilization rate, numbers of follicles and ovulations, and total and transferable embryos did not differ (P>0.05) among dietary groups. Sixty-one transferable embryos were stained and total blastomere number determined. Blastomere number was affected by diet (P<0.01); without regard to stage of development, embryos collected from cows fed SAT had lower (P<0.01) blastomere numbers (mean+/-S.E.M.; 77.1+/-3.9) than those from cows fed FLX (93.4+/-3.3) or SUN (97.2+/-3.5). Differences were most evident in the expanded blastocyst stage; at this stage, embryos of cows fed FLX and SUN diets had more blastomeres (P<0.02) than those of cows fed SAT (115.4+/-6.3, 132.3+/-8.3, and 89.3+/-9.6 cells, respectively). Although our hypothesis was only partially supported, embryonic development was enhanced in Holstein cows fed unsaturated fatty acids compared to those fed saturated fatty acids.     

The mitogen-activated protein kinase cascade MKK3-MPK6 is an important part of the jasmonate signal transduction pathway in Arabidopsis

The plant hormone jasmonic acid (JA) plays a key role in the environmental stress responses and developmental processes of plants. Although ATMYC2/JASMONATE-INSENSITIVE1 (JIN1) is a major positive regulator of JA-inducible gene expression and essential for JA-dependent developmental processes in Arabidopsis thaliana, molecular mechanisms underlying the control of ATMYC2/JIN1 expression remain largely unknown. Here, we identify a mitogen-activated protein kinase (MAPK) cascade, MAPK KINASE 3 (MKK3)-MAPK 6 (MPK6), which is activated by JA in Arabidopsis. We also show that JA negatively controls ATMYC2/JIN1 expression, based on quantitative RT-PCR and genetic analyses using gain-of-function and loss-of-function mutants of the MKK3-MPK6 cascade. These results indicate that this kinase unit plays a key role in JA-dependent negative regulation of ATMYC2/JIN1 expression. Both positive and negative regulation by JA may be used to fine-tune ATMYC2/JIN1 expression to control JA signaling. Moreover, JA-regulated root growth inhibition is affected by mutations in the MKK3-MPK6 cascade, which indicates important roles in JA signaling. We provide a model explaining how MPK6 can convert three distinct signals - JA, pathogen, and cold/salt stress - into three different sets of responses in Arabidopsis.     

Phylogenetic relationships of click beetles (Coleoptera: Elateridae) inferred from 28S ribosomal DNA: insights into the evolution of bioluminescence in Elateridae

Although the taxonomy of click beetles (family Elateridae) has been studied extensively, inconsistencies remain. We examine here the relationships between species of Elateridae based on partial sequences of nuclear 28S ribosomal DNA. Specimens were collected primarily from Japan, while luminous click beetles were also sampled from Central and South America to investigate the origins of bioluminescence in Elateridae. Neighbor-joining, maximum-parsimony, and maximum-likelihood analyses produced a consistent basal topology with high statistical support that is partially congruent with the results of previous investigations based on the morphological characteristics of larvae and adults. The most parsimonious reconstruction of the "luminous" and "nonluminous" states, based on the present molecular phylogeny, indicates that the ancestral state of Elateridae was nonluminous. This suggests that the bioluminescence in click beetle evolved independent of that of other luminous beetles, such as Lampyridae, despite their common mechanisms of bioluminescence.     

Mechanistic analysis of the phosphonate transition-state analogue-derived catalytic and non-catalytic antibody

The esterolytic catalytic antibody (catAb) has the positive charged region interacting with the carbonyl group of the ester substrate. To examine how such a region interacts with the substrate, we compared the catAb with the non-catalytic antibody (non-catAb) for interaction with the non-cleavable amide substrate (a mimic of the ester substrate) and the two end products. Surface plasmon resonance (SPR) analysis revealed that the amide substrate gave the equivalent K(d) values for the two antibodies, whereas both the on-rate and off-rate of the catAb were five-times lower than those of the non-catAb. In agreement with SPR analysis, saturation transfer difference (STD) NMR spectroscopy detected the STD signals only between the catAb and one of the product, suggesting the slower exchange rates of the amide substrate in the catAb as compared with the mixing times, whereas it was not the case with the non-catAb. Transferred nuclear Overhauser effect NMR spectroscopy showed the negative signals for only between the non-catAb and the amide substrate or the product, again suggesting the lower off-rates of the catAb as compared with the mixing times. The decreased interaction rates should be the primary consequence of the positively charged region in the combining site in the catAb.     

Essential, completely conserved glycine residue in the domain III S2-S3 linker of voltage-gated calcium channel alpha1 subunits in yeast and mammals

Voltage-gated Ca2+ channels (VGCCs) mediate the influx of Ca2+ that regulates many cellular events, and mutations in VGCC genes cause serious hereditary diseases in mammals. The yeast Saccharomyces cerevisiae has only one gene encoding the putative pore-forming alpha1 subunit of VGCC, CCH1. Here, we identify a cch1 allele producing a completely nonfunctional Cch1 protein with a Gly1265 to Glu substitution present in the domain III S2-S3 cytoplasmic linker. Comparison of amino acid sequences of this linker among 58 VGCC alpha1 subunits from 17 species reveals that a Gly residue whose position corresponds to that of the Cch1 Gly1265 is completely conserved from yeasts to humans. Systematic amino acid substitution analysis using 10 amino acids with different chemical and structural properties indicates that the Gly1265 is essential for Cch1 function because of the smallest residue volume. Replacement of the Gly959 residue of a rat brain Cav1.2 alpha1 subunit (rbCII), positionally corresponding to the yeast Cch1 Gly1265, with Glu, Ser, Lys, or Ala results in the loss of Ba2+ currents, as revealed by the patch clamp method. These results suggest that the Gly residue in the domain III S2-S3 linker is functionally indispensable from yeasts to mammals. Because the Gly residue has never been studied in any VGCC, these findings provide new insights into the structure-function relationships of VGCCs.     

Dual assemblies of an activating immune receptor, MAIR-II, with ITAM-bearing adapters DAP12 and FcRgamma chain on peritoneal macrophages

Certain activating immune receptors expressed on myeloid cells noncovalently associate with either DAP12 or FcepsilonRIgamma (FcRgamma chain), the ITAM-bearing transmembrane adapter proteins. An activating receptor, myeloid-associated Ig-like receptor (MAIR) II, is expressed on a subset of B cells and macrophages in the spleen and peritoneal cavity of mice and associates with DAP12 in these cells. However, we demonstrate here that cross-linking MAIR-II with mAb induced secretion of a significant amount of the inflammatory cytokines TNF-alpha and IL-6 from DAP12(-/-) as well as wild-type (WT) peritoneal macrophages. We show that MAIR-II associates with not only DAP12 but also FcRgamma chain homodimers in peritoneal macrophages. LPS enhanced the FcRgamma chain expression and FcRgamma chain-dependent cell surface expression of MAIR-II and had additive effects on MAIR-II-mediated inflammatory cytokine secretion from peritoneal macrophages. The lysine residue in the transmembrane region of MAIR-II was involved in the association with FcRgamma chain as well as DAP12. Our findings present the first case of an activating receptor that uses either DAP12 or FcRgamma chain as a signaling adapter. The FcRgamma chain may provide cooperation with and/or compensation for DAP12 in MAIR-II-mediated inflammatory responses by peritoneal macrophages.     

TNF-alpha drives human CD14+ monocytes to differentiate into CD70+ dendritic cells evoking Th1 and Th17 responses

Many mechanisms involving TNF-alpha, Th1 responses, and Th17 responses are implicated in chronic inflammatory autoimmune disease. Recently, the clinical impact of anti-TNF therapy on disease progression has resulted in re-evaluation of the central role of this cytokine and engendered novel concept of TNF-dependent immunity. However, the overall relationship of TNF-alpha to pathogenesis is unclear. Here, we demonstrate a TNF-dependent differentiation pathway of dendritic cells (DC) evoking Th1 and Th17 responses. CD14(+) monocytes cultured in the presence of TNF-alpha and GM-CSF converted to CD14(+) CD1a(low) adherent cells with little capacity to stimulate T cells. On stimulation by LPS, however, they produced high levels of TNF-alpha, matrix metalloproteinase (MMP)-9, and IL-23 and differentiated either into mature DC or activated macrophages (M phi). The mature DC (CD83(+) CD70(+) HLA-DR (high) CD14(low)) expressed high levels of mRNA for IL-6, IL-15, and IL-23, induced naive CD4 T cells to produce IFN-gamma and TNF-alpha, and stimulated resting CD4 T cells to secret IL-17. Intriguingly, TNF-alpha added to the monocyte culture medium determined the magnitude of LPS-induced maturation and the functions of the derived DC. In contrast, the M phi (CD14(high)CD70(+)CD83(-)HLA-DR(-)) produced large amounts of MMP-9 and TNF-alpha without exogenous TNF stimulation. These results suggest that the TNF priming of monocytes controls Th1 and Th17 responses induced by mature DC, but not inflammation induced by activated M phi. Therefore, additional stimulation of monocytes with TNF-alpha may facilitate TNF-dependent adaptive immunity together with GM-CSF-stimulated M phi-mediated innate immunity.     

Envelope Interactions in Multi-Channel Amplitude Modulation Frequency Discrimination by Cochlear Implant Users

Rationale

Previous cochlear implant (CI) studies have shown that single-channel amplitude modulation frequency discrimination (AMFD) can be improved when coherent modulation is delivered to additional channels. It is unclear whether the multi-channel advantage is due to increased loudness, multiple envelope representations, or to component channels with better temporal processing. Measuring envelope interference may shed light on how modulated channels can be combined.

Methods

In this study, multi-channel AMFD was measured in CI subjects using a 3-alternative forced-choice, non-adaptive procedure (“which interval is different?”). For the reference stimulus, the reference AM (100 Hz) was delivered to all 3 channels. For the probe stimulus, the target AM (101, 102, 104, 108, 116, 132, 164, 228, or 256 Hz) was delivered to 1 of 3 channels, and the reference AM (100 Hz) delivered to the other 2 channels. The spacing between electrodes was varied to be wide or narrow to test different degrees of channel interaction.

Results

Results showed that CI subjects were highly sensitive to interactions between the reference and target envelopes. However, performance was non-monotonic as a function of target AM frequency. For the wide spacing, there was significantly less envelope interaction when the target AM was delivered to the basal channel. For the narrow spacing, there was no effect of target AM channel. The present data were also compared to a related previous study in which the target AM was delivered to a single channel or to all 3 channels. AMFD was much better with multiple than with single channels whether the target AM was delivered to 1 of 3 or to all 3 channels. For very small differences between the reference and target AM frequencies (2–4 Hz), there was often greater sensitivity when the target AM was delivered to 1 of 3 channels versus all 3 channels, especially for narrowly spaced electrodes.

Conclusions

Besides the increased loudness, the present results also suggest that multiple envelope representations may contribute to the multi-channel advantage observed in previous AMFD studies. The different patterns of results for the wide and narrow spacing suggest a peripheral contribution to multi-channel temporal processing. Because the effect of target AM frequency was non-monotonic in this study, adaptive procedures may not be suitable to measure AMFD thresholds with interfering envelopes. Envelope interactions among multiple channels may be quite complex, depending on the envelope information presented to each channel and the relative independence of the stimulated channels.