Biomedical subjects

Nina H Pipalia

Publications and source records attributed to Nina H Pipalia.

4 recordsLinked to original sources

PubMed2006

Sterol, protein and lipid trafficking in Chinese hamster ovary cells with Niemann-Pick type C1 defect.

We studied the trafficking of sterols, lipids and proteins in Niemann-Pick type C (NPC) cells. The NPC is an inherited disorder involving the accumulation of sterol and lipids in modified late-endosome/lysosome-like storage organelles. Most sterol accumulation studies in NPC cells have been carried out using low-density lipoprotein (LDL) as the sterol source, and it has been shown that sterol efflux from late endosomes is impaired in NPC cells. In this study, we used a fluorescent sterol analog, dehydroergosterol, which can be quickly and efficiently delivered to the plasma membrane. Thus, we were able to study the trafficking kinetics of the non-LDL-derived sterol pool, and we found that dehydroergosterol accumulates in the storage organelles over the course of several hours in NPC cells. We also found that dialkylindocarbocyanine lipid-mimetic analogs that recycle efficiently from early endosomes in wild-type cells are targeted to late endosomal organelles in NPC cells, and transferrin receptors recycle slowly and inefficiently in NPC cells. These data are consistent with multiple trafficking defects in both early and late endosomes in NPC cells.

Animals↗

PubMed2006

Parallel analysis of v-Src mutant protein function using reverse transfection cell arrays.

The conversion of the genomic information produced by the recent sequencing projects into a comprehensive understanding of the human proteome has yet to occur. A new technology that represents a potential bridge between genomics and proteomics is reverse transfection. Reverse transfection cell microarrays are produced by overlaying cDNA arrays with mammalian cells, generating localized clusters of transfected cells with each cluster overexpressing a unique protein. This miniaturized cell-based microarray format affords parallel functional analysis of thousands of cDNA constructs in a high throughput format. In this report we document the development of a co-transfection methodology for reverse transfection applications. The demonstrated high co-transfection efficiency with a "marker" plasmid encoding for GFP enables the identification of transfected cells and eliminates the need for epitope-tagged constructs in cell-based high throughput screening applications using reverse transfection. This co-transfection method was used to study in parallel the structure/function of multiple versions of the v-Src protein using automated fluorescence microscopy. The wild-type v-Src protein and four mutants having insertions or deletions in the SH2 or SH3 domains displayed high levels of tyrosine kinase activity in HEK293T cells. Three other mutated v-Src proteins, including a kinase-dead version, were shown to be defective for tyrosine kinase activity. This reverse co-transfection approach is applicable for high throughput screening of both cDNA libraries and positional scanning recombinant protein libraries.

Cell Line↗

PubMed2005

Automated microscopy screening for compounds that partially revert cholesterol accumulation in Niemann-Pick C cells.

Niemann-Pick disease type C (NPC) is an autosomal recessive genetic disorder manifested by abnormal accumulation of unesterified cholesterol and other lipids. We screened combinatorially synthesized chemical libraries to identify compounds that would partially revert cholesterol accumulation. Cultured CHO cells with NPC phenotypes (CT60 and CT43) were used for screening along with normal CHO cells as a control. We developed an automated microscopy assay based on imaging of filipin fluorescence for estimating cholesterol accumulation in lysosomal storage organelles. Our primary screen of 14,956 compounds identified 14 hit compounds that caused significant reduction in cellular cholesterol accumulation at 10 microM. We then screened a secondary library of 3,962 compounds selected based on chemical similarity to the initial hits and identified 7 compounds that demonstrated greater efficacy and lower toxicity than the original hits. These compounds are effective at concentrations of 123 nM to 3 microM in reducing the cholesterol accumulation in cells with a NPC1 phenotype.

Androstenes↗

PubMed2005

A novel mechanism for protein delivery: granzyme B undergoes electrostatic exchange from serglycin to target cells.

The molecular interaction of secreted granzyme B-serglycin complexes with target cells remains undefined. Targets exposed to double-labeled granzyme B-serglycin complexes show solely the uptake of granzyme B. An in vitro model demonstrates the exchange of the granzyme from serglycin to immobilized, sulfated glycosaminoglycans. Using a combination of cell binding and internalization assays, granzyme B was found to exchange to sulfated glycosaminoglycans and, depending on the cell type, to higher affinity sites. Apoptosis induced by purified granzyme B and cytotoxic T-cells was diminished in targets with reduced cell surface glycosaminoglycan content. A mechanism of delivery is proposed entailing electrostatic transfer of granzyme B from serglycin to cell surface proteins.

Animals↗