Heterogeneity in growth and differentiation characteristics in male and female satellite cells isolated from turkey lines with different growth rates

The effects of growth- and gender-related differences on satellite cell proliferation and differentiation were investigated using satellite cells isolated from the pectoralis major muscle of a turkey line selected for increased 16-week body weight (F-line) and its unselected randombred control (RBC2-line). Proliferation rates within the F- and RBC2-lines did not differ between sexes. The F-line male and female satellite cells when compared to the RBC2-line male and female satellite cells proliferated at a faster rate. Differentiation rates were increased for the F-line male cells compared to both the F-line female and RBC2-line male satellite cells. No difference in differentiation rate was noted within the RBC2-line satellite cells. For satellite cells from females, the RBC2-line differentiated faster than the F-line. Morphological data on myotube length and the number of nuclei per myotube supported the differentiation data in that F-line male satellite cells had the longest myotubes with the most nuclei, there was no significant difference between myotubes within the RBC2-line, and female-derived myotubes from the RBC2-line were longer than those of the F-line by 96 h of fusion. These data are suggestive of both growth- and gender- related differences in satellite cell proliferation and differentiation.     

New temperature-sensitive mutants of Saccharomyces cerevisiae affecting DNA replication

Summary We have isolated new mutants of the yeast Saccharomyces cerevisiae that are defective in mitotic DNA synthesis. This was accomplished by directly screening 1100 newly isolated temperature-sensitive yeast clones for DNA synthesis defects. Ninety-seven different mutant strains were identified. Approximately half had the fast-stop DNA synthesis phenotype; synthesis ceased quickly after shifting an asynchronous population of cells to the restrictive temperature. The other half had an intermediate-rate phenotype; synthesis continued at a reduced rate for at least 3 h at the restrictive temperature. All of the DNA synthesis mutants continued protein synthesis at the restrictivetemperature. Genetic complementation analysis of temperature-sensitive segregants of these strains defined 60 apparently new complementation groups. Thirty-five of these were associated with the fast-stop phenotype, 25 with the intermediate-rate phenotype. The fast-stop groups are likely to include many genes whose products play direct roles in mitotic S phase DNA synthesis. Some of the intermediate-rate groups may be associated with S phase as well. This mutant collection should be very useful in the identification and isolation of gene products necessary for yeast DNA synthesis, in the isolation of the genes themselves, and in further analysis of the DNA replication process in vivo.     

Hemopoietic precursor cell defects in nonanemic but stem cell-deficient W44/W44 mice

Hematopoietic stem cell deficiencies cause a severe macrocytic anemia in W/Wv mice. W44/W44 mice, on the other hand, are not anemic, but, since they accept marrow implants without prior total body irradiation, they have inherited a stem cell lesion. In an attempt to identify the aberrant stem cell(s), we have determined the concentration in W44/W44 marrow of hematopoietic precursors known to be deficient in W/Wv marrow. The in vitro erythroid burst-forming units (BFU-E), the in vivo spleen colony-forming units (CFU-S), and the cells that repopulate the erythroid compartment of stem cell-deficient mice were examined. The progenitors of 7-day bursts are dramatically reduced in W/Wv marrow but are present in normal concentrations in W44/W44 marrow. W44/W44 marrow CFU-S, unlike W/Wv, generate visible spleen colonies 10 days after injection into lethally irradiated recipients. The colonies are, however, smaller and at least 2 times less numerous than those produced from equivalent numbers of +/+ marrow. An additional defect was the inability of W44/W44 stem cells to compete with genetically marked +/+ cells during erythroid repopulation. An estimate of the number of W44/W44 stem cells needed to compete with +/+ cells was provided by enriching W44/W44 progenitors fivefold. Twice as many enriched W44/W44 marrow cells as unfractionated +/+ cells were required to replace competitor cells. This suggests that there are up to 10 times fewer stem cells somewhere in the W44/W44 erythrogenerative pathway. The data support the conclusion that an erythroid progenitor less mature than the BFU-E is one of the cells most severely affected by expression of the mutant gene.     

A forced-air ventilation system for rodent cages

A novel forced-air ventilation system for rodent cages was developed. The apparatus was operated at an air flow rate of 56 L/min when used with a 230 mm wide X 450 mm long X 165 mm deep cage. Air velocity measurements in the cage did not exceed 8 m/min at animal (rat) height. The average NH3 concentration in a cage which housed two 250 g rats was less than 0.3 ppm at the end of the third day, whereas the concentration measured in a cage without the forced-air ventilation system was 150 ppm after 3 days. Tests of the water content of soiled bedding showed the forced-air ventilation system to provide a much drier environment for the rodents.     

Characterization of Tetrahymena histone H2B variants and posttranslational populations by electron capture dissociation (ECD) Fourier transform ion cyclotron mass spectrometry (FT-ICR MS)

This work describes the nature and sequence information content of the electron capture dissociation mass spectra for the intact Tetrahymena histone H2B. Two major variants of this protein were present bearing nominal modifications of both +42 and +84 Da. This work describes identification of the nature of these two modifications. For example, using gas-phase selection and isolation of the +42-Da modified species, from a background of two H2B variants each present in six or more posttranslationally modified isoforms, we were able to determine that this +42-Da modification isoform bears trimethylation rather than acetylation. LC-CIDMS analysis was also employed on digested preparations to obtain complementary detail of the nature of site-specific posttranslational modifications. This study establishes that integration of the information from these two datasets provides a comprehensive map of posttranslational occupancy for each particular covalent assemblage selected for structural investigation.     

Links between plant and fungal communities across a deforestation chronosequence in the Amazon rainforest

Understanding the interactions among microbial communities, plant communities and soil properties following deforestation could provide insights into the long-term effects of land-use change on ecosystem functions, and may help identify approaches that promote the recovery of degraded sites. We combined high-throughput sequencing of fungal rDNA and molecular barcoding of plant roots to estimate fungal and plant community composition in soil sampled across a chronosequence of deforestation. We found significant effects of land-use change on fungal community composition, which was more closely correlated to plant community composition than to changes in soil properties or geographic distance, providing evidence for strong links between above- and below-ground communities in tropical forests.     

Quantitation of a 55K cellular protein: similar amount and instability in normal and malignant mouse cells.

Quantitative expression of a specific 55,000 (55K)-molecular-weight cellular protein was studied in two groups of mouse embryo fibroblast (clonal) cells originating from two parent clones, one of which possessed high tumorigenicity and the other of which possessed very low tumorigenicity. From the clone with low tumorigenicity, tumor lines and clones were obtained by selecting rare spontaneously transformed highly tumorigenic (mutant) cells. Cells were labeled during exponential growth for 3 h at 37 degrees C, with [35S]methionine, and the cellular 55K protein was immunoprecipitated with a monoclonal antibody and quantitated. There were low and approximately equal amounts of 55K protein in cells (clones) with both low and high tumorigenicity from both groups of cells, and there was no correlation at all between quantitative expression of 55K protein and of cellular tumorigenicity. There was approximately 10- to 20-fold more 55K protein in all simian virus 40-transformed T antigen-positive derivative clones, as shown previously. The T antigen-negative revertant tumor lines and clones obtained by an immunological in vivo selection method had low amounts of 55K protein, similar to the parent cell before simian virus 40 transformation. In all of the T antigen-negative cells, including the highly tumorigenic cells, degradation (turnover?) of the 55K protein was rapid, and a half-life of 15 to 60 min was estimated from pulse-chase experiments. In all of the T antigen-positive cells the 55K protein was stable (half-life greater than 10 h). In primary cells established from the tumors induced by highly tumorigenic cells there was a very low or no detectable amount of the 55K protein. This is in contrast to the primary cells obtained from early murine embryos in which we have reported high amounts of (stable) 55K proteins.     

The residues between the two transformation effector sites of Epstein-Barr virus latent membrane protein 1 are not critical for B-lymphocyte growth transformation

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for EBV-mediated transformation of primary B lymphocytes. LMP1 spontaneously aggregates in the plasma membrane and enables two transformation effector sites (TES1 and TES2) within the 200-amino-acid cytoplasmic carboxyl terminus to constitutively engage the tumor necrosis factor receptor (TNFR)-associated factors TRAF1, TRAF2, TRAF3, and TRAF5 and the TNFR-associated death domain proteins TRADD and RIP, thereby activating NF-kappaB and c-Jun N-terminal kinase (JNK). To investigate the importance of the 60% of the LMP1 carboxyl terminus that lies between the TES1-TRAF and TES2-TRADD and -RIP binding sites, an EBV recombinant was made that contains a specific deletion of LMP1 codons 232 to 351. Surprisingly, the deletion mutant was similar to wild-type (wt) LMP1 EBV recombinants in its efficiency in transforming primary B lymphocytes into lymphoblastoid cell lines (LCLs). Mutant and wt EBV-transformed LCLs were similarly efficient in long-term outgrowth and in regrowth after endpoint dilution. Mutant and wt LMP1 proteins were also similar in their constitutive association with TRAF1, TRAF2, TRAF3, TRADD, and RIP. Mutant and wt EBV-transformed LCLs were similar in steady-state levels of Bcl2, JNK, and activated JNK proteins. The wt phenotype of recombinants with LMP1 codons 232 to 351 deleted further demarcates TES1 and TES2, underscores their central importance in B-lymphocyte growth transformation, and provides a new perspective on LMP1 sequence variation between TES1 and TES2.