Direct Comparisons of 2D and 3D Dental Microwear Proxies in Extant Herbivorous and Carnivorous Mammals

The analysis of dental microwear is commonly used by paleontologists and anthropologists to clarify the diets of extinct species, including herbivorous and carnivorous mammals. Currently, there are numerous methods employed to quantify dental microwear, varying in the types of microscopes used, magnifications, and the characterization of wear in both two dimensions and three dimensions. Results from dental microwear studies utilizing different methods are not directly comparable and human quantification of wear features (e.g., pits and scratches) introduces interobserver error, with higher error being produced by less experienced individuals. Dental microwear texture analysis (DMTA), which analyzes microwear features in three dimensions, alleviates some of the problems surrounding two-dimensional microwear methods by reducing observer bias. Here, we assess the accuracy and comparability within and between 2D and 3D dental microwear analyses in herbivorous and carnivorous mammals at the same magnification. Specifically, we compare observer-generated 2D microwear data from photosimulations of the identical scanned areas of DMTA in extant African bovids and carnivorans using a scanning white light confocal microscope at 100x magnification. Using this magnification, dental microwear features quantified in 2D were able to separate grazing and frugivorous bovids using scratch frequency; however, DMTA variables were better able to discriminate between disparate dietary niches in both carnivorous and herbivorous mammals. Further, results demonstrate significant interobserver differences in 2D microwear data, with the microwear index remaining the least variable between experienced observers, consistent with prior research. Overall, our results highlight the importance of reducing observer error and analyzing dental microwear in three dimensions in order to consistently interpret diets accurately.     

Formation of isoaspartate at two distinct sites during in vitro aging of human growth hormone

In vitro aging at pH 7.4, 37 degrees C causes natural sequence recombinant human growth hormone (rhGH), methionyl rhGH, and human pituitary growth hormone to become substrates for bovine brain protein carboxyl methyltransferase, an enzyme that modifies the "side chain" alpha-carboxyl group present at atypical isoaspartyl linkages. The substrate capacity of rhGH increased at a rate of 1.8 methyl-accepting sites/day/100 molecules of hormone. Reversed-phase high performance liquid chromatography (HPLC) of trypsin digests of aged rhGH revealed two altered peptides not present in digests of control rhGH. These two fragments, which had the amino acid compositions of residues 128-134 (Leu-Glu-Asp-Gly-Ser-Pro-Arg) and 146-158 (Phe-Asp-Thr-Asn-Ser-His-Asn-Asp-Asp-Ala-Leu-Leu-Lys), contained the majority of the induced methylation sites, 22 and 58%, respectively. Isoaspartate can result from deamidation of asparagine or isomerization of aspartate. Isomerization of Asp-130, the only candidate site in 128-134, was corroborated by coelution of the altered fragment with the synthetic isoaspartyl peptide upon reversed-phase HPLC. Evidence is presented that the altered 146-158 fragment is a mixture of two peptides resulting from deamidation of Asn-149 to form 70-80% isoaspartate and 20-30% aspartate at this position. The position of isoaspartate in the altered 146-158 fragment was deduced from mass spectrometry, which indicated a single deamidated asparagine; from methylation stoichiometry, which indicated only one methylation site; and from automated Edman degradation, which showed an absence of asparagine and a low yield of aspartate at position 149. These results show that isoaspartate formation from both aspartate and asparagine is a significant, and possibly the major, source of spontaneous covalent alteration of rhGH and that enzymatic carboxyl methylation provides a powerful tool for assessing this type of modification.     

Global Brain Ischemia and Reperfusion: Modifications in Eukaryotic Initiation Factors Associated with Inhibition of Translation Initiation

Abstract: We used in vitro translation and antibodies against phosphoserine and the eukaryotic initiation factors eIF-4E, eIF-4G, and eIF-2α to examine the effects of global brain ischemia and reperfusion on translation initiation and its regulation in a rat model of 10 min of cardiac arrest followed by resuscitation and 90 min of reperfusion. Translation reactions were performed on postmitochondrial supernatants from brain homogenates with and without aurintricarboxylic acid to separate incorporation due to run-off from incorporation due to peptide synthesis initiated in vitro. The rate of leucine incorporation due to in vitro-initiated protein synthesis in normal forebrain homogenates was ∼0.4 fmol of leucine/min/µg of protein and was unaffected by 10 min of cardiac arrest, but 90 min of reperfusion reduced this rate 83%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blots of these homogenates showed that neither 10 min of global brain ischemia nor 90 min of reperfusion induced significant alterations in the quantity or serine phosphorylation of eIF-4E. However, we observed in all 90-min-reperfused samples eIF-4G fragments that also bound eIF-4E. The amount of eIF-2α was not altered by ischemia or reperfusion, and immunoblotting after isoelectric focusing did not detect serine-phosphorylated eIF-2α in normal samples or in those obtained after ischemia without reperfusion. However, serine-phosphorylated eIF-2α was uniformly present after 90 min of reperfusion and represented 24 ± 3% of the eIF-2α in these samples. The serine phosphorylation of eIF-2α and partial fragmentation of eIF-4G observed after 90 min of reperfusion offer an explanation for the inhibition of protein synthesis.     

Tissue plasminogen activator has an O-linked fucose attached to threonine-61 in the epidermal growth factor domain

An unusual type of glycosylation has been observed for tissue plasminogen activator (t-PA). The monosaccharide fucose is glycosidically linked to threonine-61 in the epidermal growth factor region of t-PA. The presence of O-linked fucose was demonstrated by carbohydrate analysis and mass spectrometry of tryptic and chymotryptic peptides that contain this site. The susceptibility of the fucose residue to alpha-fucosidase indicated that it was in the alpha-anomeric configuration. Fucosylation of threonine-61 was observed in t-PA isolated from the Bowes melanoma cell line and from recombinant expression systems using Chinese hamster ovary or human embryonic kidney cells. Fucosylation of the homologous residue in prourokinase has also been reported recently. Our results indicate that this novel type of glycosylation may be common to the epidermal growth factor domains found in coagulation and fibrinolytic proteins and, therefore, suggest that the modification may have functional significance.     

Host-Pathogen Interactions : XX. BIOLOGICAL VARIATION IN THE PROTECTION OF SOYBEANS FROM INFECTION BY PHYTOPHTHORA MEGASPERMA F. SP. GLYCINEA

Phytophthora megasperma f.sp. glycinea, which causes soybean (Glycine max) root and stem rot, exists as several races which differ in their ability to infect a range of soybean cultivars. A glycoprotein-rich fraction (Fraction I) isolated from fungal culture fluid protects soybean seedlings from infection with compatible races. In an early study (13), seedlings were protected only by Fraction I purified from incompatible races. In 1979, seedlings were better protected by Fraction I isolated from incompatible races than by Fraction I isolated from compatible races. In 1980, seedlings were protected equally well by Fraction I from incompatible and compatible races. Materials similar in composition to Fraction I did not protect seedlings from infection. No cause could be identified for the apparent change, during the 3-year period, in the race specificity of the protection assay. Variability in the bioassay prohibited further purification or characterization of Fraction I components that protect seedlings from infection.     

Prefrontal deep projection neurons enable cognitive flexibility via persistent feedback monitoring

1. Download : Download high-res image (152KB)
2. Download : Download full-size image

     

Teratoma formation by human embryonic stem cells: Evaluation of essential parameters for future safety studies

Transplantation of human embryonic stem cells (hESC) into immune-deficient mice leads to the formation of differentiated tumors comprising all three germ layers, resembling spontaneous human teratomas. Teratoma assays are considered the gold standard for demonstrating differentiation potential of pluripotent hESC and hold promise as a standard for assessing safety among hESC-derived cell populations intended for therapeutic applications. We tested the potency of teratoma formation in seven anatomical transplantation locations (kidney capsule, muscle, subcutaneous space, peritoneal cavity, testis, liver, epididymal fat pad) in SCID mice with and without addition of Matrigel, and found that intramuscular teratoma formation was the most experimentally convenient, reproducible, and quantifiable. In the same experimental setting, we compared undifferentiated hESC and differentiated populations enriched for either beating cardiomyocytes or definitive endoderm derivatives (insulin-secreting beta cells), and showed that all cell preparations rapidly formed teratomas with varying percentages of mesoderm, ectoderm, and endoderm. In limiting dilution experiments, we found that as little as two hESC colonies spiked into feeder fibroblasts produced a teratoma, while a more rigorous single-cell titration achieved a detection limit of 1/4000. In summary, we established core parameters essential for facilitating safety profiling of hESC-derived products for future therapeutic applications.