Osteoderm histology of the Pampatheriidae (Cingulata,Xenarthra, Mammalia): Implications for systematics,osteoderm growth,and biomechanical adaptation

Pampatheres are extinct, large‐bodied cingulates, which share morphological characters with both armadillos and glyptodonts but are considered to be more closely related to the latter. The osteoderm histology of six pampathere taxa was examined and compared to the histology of other cingulate osteoderms. This study investigates the development and functional adaptation of pampathere osteoderms as well as the phylogenetic relationships of the Pampatheriidae within the Cingulata. We found that pampathere osteoderms share a uniform histological organization based on a basic diploe‐like structure. After initial stages of intramembranous growth, metaplastic ossification, that is, the direct incorporation and mineralization of pre‐existing protein fibers, plays an important role in osteoderm development and provides information on various kinds of soft tissue otherwise not preserved. The latest stages of osteoderm growth are dominated by periosteal bone formation especially in the superficial cortex. Movable band osteoderms show regular arrangements of incorporated fibers that may increase the resistance of particularly weak areas against strain. The histological composition of pampathere osteoderms is plesiomorphic in its basic structure but shows a number of derived features. A unique array of Sharpey's fibers that are incorporated into the bone matrix at sutured osteoderm margins is interpreted as a synapomorphy of pampatheres. The arrangement of dermal fibers in the deep and superficial cortexes supports the close relationship between pampatheres and glyptodonts. J. Morphol., 2012. © 2011 Wiley Periodicals, Inc.     

Development of Eye Position Dependency of Slow Phase Velocity during Caloric Stimulation

The nystagmus in patients with vestibular disorders often has an eye position dependency, called Alexander’s law, where the slow phase velocity is higher with gaze in the fast phase direction compared with gaze in the slow phase direction. Alexander’s law has been hypothesized to arise either due to adaptive changes in the velocity-to-position neural integrator, or as a consequence of processing of the vestibular-ocular reflex. We tested whether Alexander’s law arises only as a consequence of non-physiologic vestibular stimulation. We measured the time course of the development of Alexander’s law in healthy humans with nystagmus caused by three types of caloric vestibular stimulation: cold (unilateral inhibition), warm (unilateral excitation), and simultaneous bilateral bithermal (one side cold, the other warm) stimulation, mimicking the normal push-pull pattern of vestibular stimulation. Alexander’s law, measured as a negative slope of the velocity versus position curve, was observed in all conditions. A reversed pattern of eye position dependency (positive slope) was found <10% of the time. The slope often changed with nystagmus velocity (cross-correlation of nystagmus speed and slope was significant in 50% of cases), and the average lag of the slope with the speed was not significantly different from zero. Our results do not support the hypothesis that Alexander’s law can only be observed with non-physiologic vestibular stimulation. Further, the rapid development of Alexander’s law, while possible for an adaptive mechanism, is nonetheless quite fast compared to most other ocular motor adaptations. These results suggest that Alexander’s law may not be a consequence of a true adaptive mechanism.     

Electrofusion parameters for mouse two-cell embryos

We studied electrofusion of mouse two-cell embryos in order to define parameters which would result in a high yield of fused embryos. Various cell alignment times (from <10 to >60 s) and alternating current percentages (2 to 100%) were examined. The fusion parameters tested were the number of fusion pulses (1-9), pulse length (30-90 mus) and pulse strength (0.50-1.79 kV/cm). Furthermore different combinations of these three parameters were tested. In addition the influence of several embryo culture media on the fusion rates was examined. The results show that the fusion rate of the embryos increases with shorter alignment and higher percentages of the alternating current. The highest fusion rate (95%) was obtained by use of one pulse with a duration of 70 mus and a field strength of 0.60-0.79 kV/cm. The survival rate of the embryos was best if Whitten Medium was used before and after the fusion pulses. The fusion of two-cell stages results in tetraploid embryos which can serve as models for studies in polyploid cells.     

Metallothionein induction by nonsteroidal antiinflammatory drugs

In the present study we report on the effects of commonly used nonsteroidal antiinflammatory drugs on metallothionein (MT) and MT-I mRNA levels. A single dose of chloroquine (100 mg/kg), diclofenac (100 mg/kg), indomethacin (10 mg/kg), or piroxicam (100 mg/kg) was administered ip to C57B1 mice. After 18 h, MT levels were determined with a Cd-saturation radioassay. MT-I mRNA levels were measured by Northern Blot analyses using a probe containing the mouse MT-I gene. All drugs tested caused an increase in the MT content of the liver but not of the kidneys and lung. The lowest and highest effects were observed with chloroquine (8 times the control value) and diclofenac (18 times), respectively. In accordance with the stimulation of MT synthesis, increased accumulation of hepatic MT-I mRNA could be demonstrated. These results indicate that elevated MT levels may contribute to the effectiveness of nonsteroidal antiinflammatory drugs in the treatment of rheumatoid arthritis (RA).     

Genetic studies on nucleolus organizer regions (NORs) in cattle

Experimentally produced monozygotic twins, natural opposite sex blood chimeras (freemartins), and several pedigrees were used to evaluate the genetic influences on the nucleolus organizer region (NOR) patterns in cattle. In monozygotic twins, the NOR patterns of both twins are extremely similar. In chimeras, NOR patterns of genetically identical, peripheral blood lymphocytes (PBL) from the two partners resemble each other. In contrast, genetically different PBL (sib organ) differ significantly in the same environment. A high heritability of the individual NOR patterns is also demonstrated in our 23 pedigrees. In conclusion, our data demonstrate that variation in NOR expression is predominantly due to genetic factors.     

Determination of C distribution in photosynthetic serine and phosphoglycerate from grape leaves

Serine and phosphoglyceric acid are the classical marker intermediates of photorespiration and reductive carbon assimilation in C₃ plants. The present paper introduces a new and fast method for the determination of ¹⁴C distribution in these compounds by selective elimination of C-3 (NaIO₄) or C-1 (ninhydrin/ceric sulfate). Reproducibility of the procedure was found to be better than ±1% upon degradation of [U-¹⁴C]serine and [U-¹⁴C]glycerate standards.     

Purification and characterization of fructan: fructan fructosyl transferase from chicory (Cichorium intybus L.) roots

Fructan: fructan fructosyl transferase (FFT, EC 2.4.1.100) was purified from chicory (Cichorium intybus L. var. foliosum cv. Flash) roots by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, and anion- and cation-exchange chromatography. This protocol produced a 60-fold purification and a specific activity of 14.5 mol·(mg protein) ^–1·min^–1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometry, 52-kDa and 17-kDa fragments were found, suggesting that the enzyme was a heterodimer. Optimal activity was found between pH 5.5 and 6.5. The enzyme used 1-kestose, 1,1-nystose, oligofructan and commercial chicory root inulin (degree of polymerization 10) as donors and acceptors. Sucrose was the best acceptor but could not be used as a donor. However, at higher concentrations sucrose acted as a competitive inhibitor for donors of FFT. 1-Kestose was the most efficient and 1,1-nystose the least efficient donor. The purified enzyme exhibited -fructosidase activity, specially at higher temperatures and lower substrate concentrations. The synthesis of fructans from 1-kestose decreased at higher temperatures (5–50°C). Therefore enzyme assays were performed at 0°C. The same fructan oligosaccharides, with a distribution similar to that observed in vivo, were obtained upon incubation of the enzyme with sucrose and commercial chicory root inulin.Abbreviations Con A concanavalin A - DP degree of polymerization - FFT fructan: fructan fructosyl transferase - Fru fructose - Glc glucose - Kes 1-kestose - MALDI-TOF MS matrix-assisted laser desorption ionisation time of flight mass spectrometry - Nys 1,1-nystose - pI isoelectric point - SST sucrose: sucrose fructosyl transferase - Suc sucrose The authors would like to thank E. Nackaerts for valuable assistance. W. Van den Ende is also grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants. P. Verhaert is a research associate of the NFSR. This work was also supported by grant OT/91/18 from the Research Fund K.U. Leuven.     

Effects of different cryoprotectants and carbohydrates on freezing of matured and unmatured bovine oocytes

Cumulus cell-enclosed bovine oocytes in germinal vesicle (GV) and in metaphase II (MII) stages were cryopreserved. Different concentrations (1 M; 1.5 M) of various cryoprotectants (glycerol, PROH, DMSO) were tested. After thawing, the oocytes were exposed to various carbohydrates (sucrose, lactose, trehalose) at a concentration of 0.1 M and 0.25 M for cryoprotectant removal. Developmental capacity of the frozen-thawed oocytes was studied by in vitro maturation, fertilization and culture. We found no difference in subsequent development using glycerol or PROH for GV and MII oocytes. The DMSO treatment led to significantly better cleavage and development up to 4-cell stage in MII oocytes. Development beyond the 8-cell stage was obtained only when unmatured oocytes were frozen. No difference in the efficiency of the 3 cryoprotectants was detected in MII oocytes. However, in GV oocytes, glycerol and PROH yielded significantly better cleavage and 4-cell rate compared to DMSO (P<0.001). Influence of the concentration of a cryoprotectant on development was not observed in GV or MII oocytes. Among the 3 cryoprotectants, DMSO was less suitable, at both concentrations, than PROH and glycerol for the development of 6- to 8-cell stage embryos in the GV group. In the MII group, 1.5 M DMSO was as efficient as PROH and as glycerol at a 1.5-M concentration, and it was more efficient than 1 M glycerol. The use of carbohydrates during rehydration did not render a beneficial effect at either of the 2 concentrations, and when no carbohydrates were used in the MII group the oocytes cleaved better than GV oocytes.