Biomedical subjects

A M Rampalli

Publications and source records attributed to A M Rampalli.

4 recordsLinked to original sources

PubMed1999

Changes in cyclin dependent kinase expression and activity accompanying lens fiber cell differentiation.

Previous studies from this laboratory have shown that differentiating lens fiber cells contain two active cyclin dependent kinases (Cdks), Cdk1 and Cdk5. The present study was undertaken to explore the expression and regulation of six additional members of the Cdk family (Cdk2, Cdk3, Cdk4, Cdk6, Cdk7 and Cdk8) during lens differentiation. Differentiating lens fiber cells were separated from lens epithelial cells by microdissection of developing rat lenses [embryonic day 16 (E16) to postnatal day 8 (P8)] and Cdk expression was assessed by RT-PCR and immunoblotting. Two Cdks (Cdk3 and Cdk6) were not expressed in lens fiber cells or epithelial cells during this developmental period. In the lens epithelium, we detected proteins and mRNAs corresponding to all other Cdks examined (Cdk2, Cdk4, Cdk7, Cdk8) throughout this developmental period. Epithelial cells showed significant Cdk2 activity, which decreased with developmental age, but no significant activity was detected for Cdk4, Cdk7, or Cdk8. Fiber cells contained all four Cdk proteins and the corresponding Cdk mRNAs except for Cdk2 mRNA. None of the Cdks examined showed significant kinase activity in fiber cells. Immunoprecipitates of Cdk2 and Cdk4 from fiber cells contained p57(kip2), supporting the view that this Cdk inhibitor blocks the activity of these Cdks in lens fibers. In contrast, p57(kip2)did not co-immunoprecipitate with Cdk5 from lens fibers. These findings suggest that the differential affinity of p57(kip2)for members of the Cdk family may provide a mechanism for specific regulation of individual Cdks during fiber cell differentiation.

Animals↗

PubMed1998

pRb and p107 regulate E2F activity during lens fiber cell differentiation.

During growth arrest and differentiation, activity of the E2F family of transcription factors is inhibited by interactions with pRb and the related proteins, p107 and p130. To determine which members of the E2F and pRb families may contribute to growth arrest as lens epithelial cells differentiate into fiber cells, we examined the expression of individual E2F species and characterized the E2F protein complexes formed in rat lens epithelia and fibers. RT/PCR detected all five known members of the E2F family in lens epithelial cells, but only E2F-1, E2F-3, and E2F-5 in fiber cells. Proteins extracted from lens epithelia of newborn rats formed at least two specific complexes with an E2F consensus oligonucleotide. Proteins from lens fiber cells formed three specific complexes, one of which comigrated with an epithelial cell complex. Incubation of epithelial and fiber cell extracts with an antibody specific for p107 demonstrated that two fiber cell complexes and one epithelial cell complex contained p107. Although the remaining fiber cell complex did not react with antibodies to pRb or p130 in this assay, a strong reaction with pRb antibody was observed when the electromobility shifted complexes were subsequently immunoblotted (shift/Western assay). Immunocytochemistry confirmed that pRb protein is present in the nuclei of both epithelial cells and fiber cells. Immunoblotting of whole cell extracts with pRb antibody showed multiple, phosphorylated forms of pRb in the epithelial cells, but predominantly hypophosphorylated pRb in the fiber cells. None of the complexes formed with E2F were recognized exclusively by the p130 antibody, although the previously identified p107 complexes reacted weakly. The absence of p130/E2F complexes was correlated with the presence of multiple ubiquitinated forms of p130, especially in the fiber cells. Thus, although p130/E2F complexes are implicated in the terminal differentiation of many cell types, in differentiating lens fiber cells pRb and p107 seem to be the primary regulators of E2F activity.

Animals↗

PubMed1996

Expression of alternatively spliced PCTAIRE-1 mRNA in PC12 cells and neonatal rat brain.

A rat PCTAIRE-1 cDNA clone was isolated by immunoscreening of a PC12 cDNA library, followed by 5' RACE (rapid amplification of cDNA ends) to determine the 5' end. The rat PCTAIRE-1 cDNA sequence is 96% identical to mouse PCTAIRE-1 and contains an alternatively spliced exon of 131 bp near the 5' end. Although a mouse cDNA containing this exon has been reported, examination of several mouse cell lines provided no evidence for expression of the corresponding mRNA (Okuda et al., 1992). In contrast, reverse transcription and polymerase chain reaction (RT/PCR) across this region using RNA from proliferating, differentiated, and apoptotic PC12 cells demonstrated that alternatively spliced forms of PCTAIRE-1 mRNA with and without this exon are expressed. Both forms of PCTAIRE-1 mRNA are also expressed in vivo in neonatal rat brain, although other tissues examined contained only the form lacking the alternatively spliced exon. In the absence of the alternatively spliced exon PCTAIRE-1 mRNA contains an open reading frame of 1488 bp, corresponding to a 55-kDa protein that is 97% identical to mouse PCTAIRE-1 protein. When the alternatively spliced exon is present, this open reading frame is terminated by a stop codon and a second open reading frame is initiated, predicting a second PCTAIRE-1 protein of 52 kDa. The two predicted PCTAIRE-1 proteins are identical downstream of the splice site, but share no homology at their N-terminal ends.

Alternative Splicing↗

PubMed1995

Insulin regulates expression of c-fos and c-jun and suppresses apoptosis of lens epithelial cells.

This study investigates whether insulin (a differentiation factor for lens epithelial cells) acts as a survival factor. In the absence of insulin, 6-day embryonic chicken lens epithelial explants undergo apoptosis as shown by changes in cell morphology, DNA fragmentation, and loss of trypan blue exclusion. Insulin inhibits these changes and promotes survival of the cells. Aurintricarboxylic acid suppresses the apoptosis of lens explants. In contrast to 6-day embryonic explants, 19-day embryonic explants survive in the absence of insulin, presumably due to an endogenous survival factor. To explore the mechanism of the action of insulin as a survival factor for 6-day embryonic lens explants, we compared the pattern of cell cycle markers (c-fos, c-jun, c-myc, p53, histone H3, thymidine kinase, and cyclin B) in both apoptotic and differentiating lens explants. In the presence of insulin, the expression of c-fos and c-jun was down-regulated after an initial induction. Expression of these genes was also induced in the absence of insulin, but mRNA levels remained elevated as the cells underwent apoptosis. In contrast, expression of c-myc, p53, histone H3, thymidine kinase, and cyclin B showed only minor differences in differentiating and apoptotic cells. Since c-fos and c-jun have been shown to play a role in apoptosis in other cell types, the ability of insulin to regulate expression of these genes may be central to its ability to act as a survival factor for lens epithelial cells.

Animals↗