Isolation, partial chemical and biological characterization of thymone B

A new approach to the study of the molecular arrangements of proteins in membranes is described. Irradiation with visible light of native erythrocytes or washed erythrocyte membranes suspended in buffers containing a) riboflavin, fluorescein or fluorescein coupled to dextran and b) ³H-labelled tryptophan resulted in incorporation of radioactivity into the membrane proteins. Polyacrylamide gel electrophoresis of solubilized membranes followed by radioactivity measurements of the separated membrane proteins revealed that in native erythrocytes the protein components known to be located at the exterior cell surface, Band 3 and the major sialoglycoproteins became specifically labelled, whereas in washed lysed cells all of the major membrane proteins were labelled.     

Postpartum reproductive performance in the multiparous mare fed to obesity

Twenty multiparous Quarter Horse mares were assigned to one of two treatment groups at 40 to 75 d of pregnancy. Group 1 was the control group and the mares were fed to maintain a moderate degree of body fat (condition score 5.5 to 7). Group 2 was the obese group and the mares were fed to achieve (prepartum) and then maintain (post partum) an extremely high degree of body fat (condition score 9). Estrous intensity was evaluated using subjective teasing scores ranging from 0 (rejection) to 4 (maximum receptivity). Mares were artificially inseminated beginning with the second postpartum ovulatory cycle; the study was terminated after 63 d of pregnancy. Duration of estrus, maximum teasing score and the number of mares exhibiting overt estrus (teasing score > 2) did not differ between treatment groups during the first and second postpartum ovulatory cycles. The intervals from foaling to first cycle ovulation, foaling to second cycle ovulation, and first to second cycle ovulation were also similar between treatment groups. All mares in both treatment groups conceived and maintained pregnancy. The first cycle conception rate and the number of cycles per conception did not differ between treatment groups. A high degree of body fat produced by overfeeding during gestation did not adversely affect postpartum reproductive performance in the multiparous mare.     

Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.     

Continuous,high capacity reconstitution of nutrient media from concentrated intermediates

We designed an Integrated Media Preparation System (IMPS) for continuous, on-line preparation of cell culture media and delivery to intermediate storage vessels or directly to a bioreactor. Key components of the IMPS include: a high precision, continuous fluid mixing device; formulation-specific liquid medium concentrates; validated process controls and membrane filtration; and automated dispensing into large volume flexible plastic containers. The IMPS system is designed to produce sterile, single-strength liquid medium from common raw materials at a delivery rate of 1000–3000 liters per hour and will manufacture homogenous batches from several thousand liters to over 60,000 liters. Fortified nutrient media prepared from multi-component 50X concentrates have been demonstrated to accelerate bioreactor seed chains, increase product yield, and reduce the overall manufacturing cost of nutrient medium. A productivity matrix will analyze the fully-loaded costs and contrast alternative methods for media preparation against projected biological yield.Abbreviations IMPS Integrated Media Preparation System - 50X Nutrient fluid components formulated at fifty-fold final use concentration - 1X Nutrient fluid formulated at final, single-strength use concentration - cGMP Current Good Manufacturing Practices - SCADA Supervisory Control and Data Acquisition - PLC Process Logic Controller - LTI Life Technologies, Inc. - WFI Water for Injection - CIP Clean in place - SIP Sterilize in place - HPLC High performance liquid chromatography - DMEM Dulbecco's Modified Eagle's Medium     

Mouse oocyte maturation is affected by lithium via the polyphosphoinositide metabolism and the microtubule network

The incubation of mechanically denuded mouse oocytes in medium containing LiCl delayed both germinal vesicle breakdown (GVBD) and polar body extrusion in a dose-dependent and reversible manner. When myo-inositol alone was added to the culture medium, we observed that it accelerated GVBD and increased the rate of polar body extrusion, whereas, when combined with LiCl, the normal timing of GVBD was recovered. In the same way, when inositol trisphosphate (InsP₃) was microinjected into the ooplasma, we observed an important improvement of the rate of GVBD, as compared to control oocytes, and prevention of lithium inhibition. However, neither myo-inositol nor InsP₃ were able to rescue totally the oocytes from the negative effect of lithium on polar body extrusion. Moreover, lithium induced some important changes in microtubule and chromosome organizations. Before extrusion of the first polar body, the reduction of the spindle size or the appearance of short individualized chromosomes dispersed around a large aster of microtubules were often observed, whereas, after polar body extrusion, the spindle appeared smaller and chromosomes were often trapped in the midbody. Thus lithium affects mouse oocyte maturation at two different levels: GVBD and polar body extrusion. Whereas the former seems to be affected via polyphosphoinositide turnover, the latter is InsP₃-independent and seems to be influenced negatively via underdevelopment of microtubular structures. © 1994 Wiley-Liss, Inc.     

The expression of nuclear lamin A and C epitopes is regulated by the developmental stage of the cytoplasm in mouse oocytes or embryos.

The cytoplasmic regulation of changes of nuclear lamin antigens was examined by transferring 16-cell stage blastomeres into mouse oocytes. Sixteen-cell stage blastomeres were transferred to either pronuclear eggs, enucleated pronuclear eggs or metaphase II oocytes, which were subsequently activated. Pronuclei react with a monoclonal antibody to A/C lamins (J9), whereas nuclei from 16-cell stage blastomeres do not react with J9. However, after transfer of 16-cell stage nuclei to activated metaphase II oocytes, the transferred nuclei acquire the antigen. This is in contrast to 16-cell nuclei that were transferred to intact or enucleated pronuclear eggs; i.e., the nuclei only faintly acquired the A/C epitope. These results suggest that the developmental stage of the cytoplasm regulates the exposure of nuclear lamina epitopes, perhaps by limiting the supply of lamin A/C in the oocyte or because nuclear lamina assembly can only occur at the telophase transition. Furthermore, it appears that there is some exchange of the A/C epitope between (pro)nuclei within the same cell but that the majority of the A/C lamin epitope can be removed from a cell with (pro)nuclear removal.     

c-mos proto-oncogene product is partly degraded after release from meiotic arrest and persists during interphase in mouse zygotes.

Recently, it has been shown that the product of the c-mos proto-oncogene is a component of cytostatic factor, an activity present in unfertilized eggs from vertebrates that arrests the cell cycle in metaphase of the second meiotic division (metaphase II) possibly by stabilizing maturation-promoting factor (MPF). We have studied the behavior of the c-mos product in metaphase II mouse oocytes and soon after activation. The amount of c-mos in the oocyte was still very high after second polar body extrusion, when cyclin B has been degraded and MPF activity had decreased dramatically. Degradation of c-mos takes place later, during the G1 phase of the first cell cycle and a residual amount of c-mos is detectable during the first zygotic interphase. Our data show that the degradation of c-mos is not involved in the release from the metaphase arrest.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Ecological specialization and niche overlap of subterranean rodents inferred from DNA metabarcoding diet analysis

Knowledge of how animal species use food resources available in the environment can increase our understanding of many ecological processes. However, obtaining this information using traditional methods is difficult for species feeding on a large variety of food items in highly diverse environments. We amplified the DNA of plants for 306 scat and 40 soil samples, and applied an environmental DNA metabarcoding approach to investigate food preferences, degree of diet specialization and diet overlap of seven herbivore rodent species of the genus Ctenomys distributed in southern and midwestern Brazil. The metabarcoding approach revealed that these species consume more than 60% of the plant families recovered in soil samples, indicating generalist feeding habits of ctenomyids. The family Poaceae was the most common food resource retrieved in scats of all species as well in soil samples. Niche overlap analysis indicated high overlap in the plant families and molecular operational taxonomic units consumed, mainly among the southern species. Interspecific differences in diet composition were influenced, among other factors, by the availability of resources in the environment. In addition, our results provide support for the hypothesis that the allopatric distributions of ctenomyids allow them to exploit the same range of resources when available, possibly because of the absence of interspecific competition.