A convenient method for a-amylase and invertase electrophoresis on cellulose acetate strips

Electrophoresis of a-amylase and invertas

DNA mediated chromatin pull-down for the study of chromatin replication

Chromatin replication involves duplicating DNA while maintaining epigenetic information. These processes are critical for genome stability and for preserving cell-type identity. Here we describe a simple experimental approach that allows chromatin to be captured and its content analysed after in vivo replication and labeling of DNA by cellular DNA polymerases. We show that this technique is highly specific and that proteins bound to the replicated DNA can be analyzed by both immunological techniques and large scale mass spectrometry. As proof of concept we have used this novel procedure to begin investigating the relationship between chromatin protein composition and the temporal programme of DNA replication in human cells. It is expected that this technique will become a widely used tool to address how chromatin proteins assemble onto newly replicated DNA after passage of a replication fork and how chromatin maturation is coupled to DNA synthesis

Attenuation by all-trans-retinoic acid of sodium chloride-enhanced gastric carcinogenesis induced by N-methyl-N′-nitro-N-nitrosoguanidine in Wistar rats

The effect of prolonged administration of all-trans-retinoic acid (RA) on sodium chloride-enhanced gastric carcinogenesis induced by N-methyl-N′-nitro-N-nitrosoguanidine, and the labelling and apoptotic indices and immunoreactivity of transforming growth factor (TGF) α in the gastric cancers was investigated in Wistar rats. After 25 weeks of carcinogen treatment, the rats were given chow pellets containing 10% sodium chloride and subcutaneous injections of RA at doses of 0.75 or 1.5 mg kg−1 body weight every other day. In week 52, oral supplementation with sodium chloride significantly increased the incidence of gastric cancers compared with the untreated controls. Long-term administration of RA at both doses significantly reduced the incidence of gastric cancers, which was enhanced by oral administration of sodium chloride. RA at both doses significantly decreased the labelling index and TGF-α immunoreactivity of gastric cancers, which were enhanced by administration of sodium chloride, and significantly increased the apoptotic index of cancers, which was lowered by administration of sodium chloride. These findings suggest that RA attenuates gastric carcinogenesis, enhanced by sodium chloride, by increasing apoptosis, decreasing DNA synthesis, and reducing TGF-α expression in gastric cancers. © 1999 Cancer Research Campaig

CREB Is Activated by Muscle Injury and Promotes Muscle Regeneration

The cAMP response element binding protein (CREB) plays key roles in differentiation of embryonic skeletal muscle progenitors and survival of adult skeletal muscle. However, little is known about the physiologic signals that activate CREB in normal muscle. Here we show that CREB phosphorylation and target genes are induced after acute muscle injury and during regeneration due to genetic mutation. Activated CREB localizes to both myogenic precursor cells and newly regenerating myofibers within regenerating areas. Moreover, we found that signals from damaged skeletal muscle tissue induce CREB phosphorylation and target gene expression in primary mouse myoblasts. An activated CREB mutant (CREBY134F) potentiates myoblast proliferation as well as expression of early myogenic transcription factors in cultured primary myocytes. Consistently, activated CREB-YF promotes myoblast proliferation after acute muscle injury in vivo and enhances muscle regeneration in dystrophic mdx mice. Our findings reveal a new physiologic function for CREB in contributing to skeletal muscle regeneration

Evidence for supporting cell mitosis in response to acoustic trauma in the avian inner ear

Acoustic overstimulation can lead to sensory cell (hair cell) loss in the auditory epithelium. Damaged hair cells in the organ of Corti (the mammalian auditory end-organ) degenerate and are replaced by non-sensory cells (supporting cells) which construct an irreversible scar. In birds, however, auditory hair cells which are damaged by acoustic trauma or ototoxic drugs may be replaced by new hair cells. As first step in determining the mechanism of hair cell regeneration, we developed an assay for cell divisions in the auditory epithelium after acoustic trauma. The results of these experiments demonstrate that supporting cells in damaged regions of the auditory epithelium incorporate the DNA-specific marker bromodeoxyuridine as early as one day after noise exposure. We provide direct evidence that following acoustic insult to the avian inner ear, supporting cells which reside within the sensory epithelium divide near the luminal surface and repopulate the epithelium. These results suggest that supporting cells participate in scar formation during hair cell degeneration, and produce new cells for regeneration.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47429/1/11068_2005_Article_BF01191727.pd

Dynein light chain 1 functions in somatic cyst cells regulate spermatogonial divisions in Drosophila

Stem cell progeny often undergo transit amplifying divisions before differentiation. In Drosophila, a spermatogonial precursor divides four times within an enclosure formed by two somatic-origin cyst cells, before differentiating into spermatocytes. Although germline and cyst cell-intrinsic factors are known to regulate these divisions, the mechanistic details are unclear. Here, we show that loss of dynein-light-chain-1 (DDLC1/LC8) in the cyst cells eliminates bag-of-marbles (bam) expression in spermatogonia, causing gonial cell hyperplasia in Drosophila testis. The phenotype is dominantly enhanced by Dhc64C (cytoplasmic Dynein) and didum (Myosin V) loss-of-function alleles. Loss of DDLC1 or Myosin V in the cyst cells also affects their differentiation. Furthermore, cyst cell-specific loss of ddlc1 disrupts Armadillo, DE-cadherin and Integrin-βPS localizations in the cyst. Together, these results suggest that Dynein and Myosin V activities, and independent DDLC1 functions in the cyst cells organize the somatic microenvironment that regulates spermatogonial proliferation and differentiation

Measuring proliferation in breast cancer: practicalities and applications

Various methods are available for the measurement of proliferation rates in tumours, including mitotic counts, estimation of the fraction of cells in S-phase of the cell cycle and immunohistochemistry of proliferation-associated antigens. The evidence, advantages and disadvantages for each of these methods along with other novel approaches is reviewed in relation to breast cancer. The potential clinical applications of proliferative indices are discussed, including their use as prognostic indicators and predictors of response to systemic therapy