Muscle regeneration during hindlimb unloading results in a reduction in muscle size after reloading.

The hindlimb-unloading model was used to study the ability of muscle injured in a weightless environment to recover after reloading. Satellite cell mitotic activity and DNA unit size were determined in injured and intact soleus muscles from hindlimb-unloaded and age-matched weight-bearing rats at the conclusion of 28 days of hindlimb unloading, 2 wk after reloading, and 9 wk after reloading. The body weights of hindlimb-unloaded rats were significantly (P < 0.05) less than those of weight-bearing rats at the conclusion of hindlimb unloading, but they were the same (P > 0.05) as those of weight-bearing rats 2 and 9 wk after reloading. The soleus muscle weight, soleus muscle weight-to-body weight ratio, myofiber diameter, number of nuclei per millimeter, and DNA unit size were significantly (P < 0.05) smaller for the injured soleus muscles from hindlimb-unloaded rats than for the soleus muscles from weight-bearing rats at each recovery time. Satellite cell mitotic activity was significantly (P < 0.05) higher in the injured soleus muscles from hindlimb-unloaded rats than from weight-bearing rats 2 wk after reloading, but it was the same (P > 0.05) as in the injured soleus muscles from weight-bearing rats 9 wk after reloading. The injured soleus muscles from hindlimb-unloaded rats failed to achieve weight-bearing muscle size 9 wk after reloading, because incomplete compensation for the decrease in myonuclear accretion and DNA unit size expansion occurred during the unloading period.     

Identification of the lacZ insertion site and beta-galactosidase expression in transgenic chickens

The quail:chick chimera system is a classical research model in developmental biology. An improvement over the quail:chick chimera system would be a line of transgenic chickens expressing a reporter gene. Transgenic chickens carrying lacZ and expressing bacterial beta-galactosidase have been generated, but complete characterization of the insertion event and characterization of beta-galactosidase expression have not previously been available. The genomic sequences flanking the retroviral insertion site have now been identified by using inverse polymerase chain reaction (PCR), homozygous individuals have been identified by using PCR-based genotyping, and beta-galactosidase expression has been evaluated by using Western analysis and histochemistry. Based upon the current draft of the chicken genome, the viral insertion carrying the lacZ gene has been located on chromosome 11 within the predicted gene for neurotactin/fractalkine (CX3CL1); neurotactin mRNA expression appears to be missing from the brain of homozygous individuals. When Generation 2 (G2) lacZ-positive individuals were inter-mated, they generated 361 G3 progeny; 82 were homozyous for lacZ (22.7%), 97 were wild-type non-transgenic (26.9%), and 182 (50.4%) were hemizygous for lacZ. Western analysis revealed the highest expression in the muscle and liver. With the identification of homozygous birds, the line of chickens is now designated NCSU-Blue1.     

Myonuclear apoptosis occurs during early posthatch starvation

Apoptosis is a naturally occurring process; it is important for the final shape and size of developing tissues, and it is characterized by some morphological features such as plasma membrane blebbing, nuclear breakdown, chromosomal fragmentation and apoptotic bodies followed by phagocytosis. The objective of the study was to evaluate the occurrence of apoptosis in chickens immediately posthatch under fed and starved conditions. Male broiler chickens were or were not provided feed for the first 3 days posthatch. Chickens were killed immediately after hatch, at 1 day of age, at 2 days of age and at 3 days of age. The Pectoralis thoracicus was removed, fixed, dehydrated, cleared and embedded in paraffin. Muscle sections were labeled using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick-End Labeling (TUNEL) for detection of apoptotic nuclei. Body weights were lower (P<0.05) in the starved compared to the fed group at 2 and 3 days posthatch. Myofiber cross-sectional area was only smaller (P<0.05) in the starved compared to the fed birds at 3 days posthatch. TUNEL-positive nuclei were present at all days for the fed and starved groups. The proportion of TUNEL-positive nuclei was higher (P<0.05) for the starved group at day 2 and day 3 posthatch compared to the fed group at 3 days posthatch. Apoptosis is a mechanism that contributes to the smaller myofiber size observed at 3 days posthatch.     

Quantitation of Satellite Cell Proliferation in Vivo Using Image Analysis

A nonisotopic, double fluorescence technique was developed to study myogenic satellite cell proliferation in posthatch turkey skeletal muscle. Labeled satellite cell nuclei were identified on enzymatically isolated myofiber segments using a mouse monoclonal antibody (anti-BrdU) followed by fluorescein-5-isothiocyanate (FITC) conjugated goat anti-mouse IgG secondary antibody. Myofiber nuclei (myonuclei + satellite cell nuclei) were counterstained with propidium iodide (PI). The myofiber segment length, myofiber segment diameter, and the number of PI and FITC labeled nuclei contained in each segment was determined using a Nikon fluorescence microscope, a SIT video camera and Image-1 software. Data collected by three different operators of the image analysis system revealed 5.0 ± 1.4 satellite cell nuclei per 1000 myofiber nuclei and 5284 ± 462 μm³ of cytoplasm surrounding each myofiber nucleus in the pectoralis thoracicus of 9-week-old tom turkeys. BrdU immunohistochemistry coupled with the new approach of PI staining of whole myofiber mounts is an effective combination to allow the use of an efficient semi-automated image analysis protocol.     

Drought‐resistant fungi control soil organic matter decomposition and its response to temperature

Microbial‐mediated decomposition of soil organic matter (SOM) ultimately makes a considerable contribution to soil respiration, which is typically the main source of CO₂ arising from terrestrial ecosystems. Despite this central role in the decomposition of SOM, few studies have been conducted on how climate change may affect the soil microbial community and, furthermore, on how possible climate‐change induced alterations in the ecology of microbial communities may affect soil CO₂ emissions. Here we present the results of a seasonal study on soil microbial community structure, SOM decomposition and its temperature sensitivity in two representative Mediterranean ecosystems where precipitation/throughfall exclusion has taken place during the last 10 years. Bacterial and fungal diversity was estimated using the terminal restriction fragment length polymorphism technique. Our results show that fungal diversity was less sensitive to seasonal changes in moisture, temperature and plant activity than bacterial diversity. On the other hand, fungal communities showed the ability to dynamically adapt throughout the seasons. Fungi also coped better with the 10 years of precipitation/throughfall exclusion compared with bacteria. The high resistance of fungal diversity to changes with respect to bacteria may open the controversy as to whether future ‘drier conditions’ for Mediterranean regions might favor fungal dominated microbial communities. Finally, our results indicate that the fungal community exerted a strong influence over the temporal and spatial variability of SOM decomposition and its sensitivity to temperature. The results, therefore, highlight the important role of fungi in the decomposition of terrestrial SOM, especially under the harsh environmental conditions of Mediterranean ecosystems, for which models predict even drier conditions in the future.     

CCN2 promotes keratinocyte adhesion and migration via integrin α5β1

Background

CCN2, (a.k.a. connective tissue growth factor and CTGF) has emerged as a regulator of cell migration. While the importance of CCN2 for the fibrotic process in wound healing has been well studied, the effect of CCN2 on keratinocyte function is not well understood. In this study, we investigated the mechanism behind CCN2-driven keratinocyte adhesion and migration.Materials and methods: Adhesion assays were performed by coating wells with 10 μg/ml fibronectin (FN) or phosphate-buffered saline (PBS). Keratinocytes were seeded in the presence or absence of 200 ng/ml CCN2, 5 mmol/l ethylenediaminetetraacetic acid, 10 mmol/l cations, 500 μl arginine–glycine–aspartic acid (RGD), 500 μM arginine–glycine–glutamate–serine (RGES), and 10 μg/ml anti-integrin blocking antibodies. Migration studies were performed using a modified Boyden chamber assay. Quantitative PCR was used to study the effect of CCN2 on integrin subunit mRNA expression. To block intracellular pathways, keratinocytes were pretreated with 20 μM PD98059 (MEK-1 inhibitor) or 20 μM PF573228 (FAK inhibitor) for 60 min prior the addition of CCN2. Western blot was used to measure CCN2, p-ERK1/2, and ERK1/2.Results: CCN2 enhanced keratinocyte adhesion to fibronectin via integrin α5β1. The addition of anti-integrin α5β1 antibodies reduced CCN2-mediated keratinocyte migration. In addition, CCN2 regulated mRNA and protein expression of integrin subunits α5 and β1. CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1-specific inhibitor PD98059 markedly reduced CCN2-induced keratinocyte migration.Conclusions: Our results demonstrate that CCN2 enhances keratinocyte adhesion and migration through integrin α5β1 and activation of the FAK-MAPK signaling cascade.     

The effect of early dietary amino acid levels on muscle satellite cell dynamics in turkeys

Understanding the relationship between nutrition and satellite cell activity will be beneficial in obtaining optimal muscle growth and meat production. The objective of this study was to evaluate the effect of early post-hatch levels of dietary amino acids+/-0.88 NRC, 1.00 NRC, and 1.12 NRC), and feed deprivation on the satellite cell mitotic activity, pectoralis thoracicus muscle weight, and body weight of male turkeys (Meleagris gallopavo). Birds from each treatment were injected with 5-bromo-2'-deoxyuridine (BrdU) to label mitotically active cells. The right pectoralis thoracicus was harvested 1 h after BrdU injection for immunohistochemical and myofiber diameter analysis. On the third day post-hatch, satellite cell mitotic activity was the highest (P<0.05) in the 0.88 NRC amino acid treatment group and the lowest (P<0.05) in the feed-deprived group. On the fourth day post-hatch, feed-deprived birds exhibited the lowest (P<0.05) satellite cell mitotic activity and muscle weight. At 140 days of age, there were no significant differences (P>0.05) between treatments in body weight or pectoralis thoracicus muscle weight. Research evaluating species-related differences in apoptotic events and in genes regulating cell proliferation may be necessary to devise feeding strategies aimed at obtaining optimal pectoralis thoracicus muscle yield at market age.     

A chicken mRNA similar to heterogeneous nuclear ribonucleoprotein H1

Heterogeneous nuclear ribonucleoproteins are predominantly nuclear RNA-binding proteins that function in a variety of cellular activities. The objective of these experiments was to clone a cDNA for a chicken protein similar to other previously reported heterogeneous ribonucleoproteins for other species. The 5' and 3' ends of the chicken mRNA were cloned using Rapid Amplification of cDNA Ends (RACE). Subsequently, the expression of the mRNA sequence was confirmed via Northern analysis. The deduced amino acid sequence was approximately 86% identical to corresponding regions of human, mouse, or zebrafish proteins similar to heterogeneous nuclear ribonucleoprotein H1. The expression data confirmed the size of the predicted mRNA sequence. The newly identified sequence may be employed in future studies aimed at understanding the role of heterogeneous nuclear ribonucleoproteins in avian species.     

Nitric oxide donors, sodium nitroprusside and S-nitroso-N-acetylpenicillamine, stimulate myoblast proliferation in vitro

Summary Nitric oxide (NO) is an inter- and intracellular messenger involved in a variety of physiologic and pathophysiologic conditions. The effect of two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) and their effect on myoblast proliferation was examined. Both donors stimulated an increase in myoblast cell number over a range (1–10 μM) of donor concentrations. However, 50 μM SNAP inhibited myoblast proliferation. Cell numbers from cultures treated with degraded 10 μM SNAP were equivalent to the control. Therefore, it appears NO can stimulate as well as inhibit myoblast proliferation.     

Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation

Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60. The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and poly-morphonuclear leukocytes (PMN) over 9 d of culture in 1.3 percent dimethylsulfoxide (DMSO). As the HL-60 cells mature, the rate of O(2-) production increase 18-fold, with a progressive shortening of the lag time required for activation. Hexosemonophosphate shunt activity rises concomitantly. Ingestin of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities. Degranulation, as measured by release of β-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective. However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of O(2-) generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100 percent that of normal PMN. DMSO- induced differentiation of HL-60 cells is a promising model for myeloid development.