Optimized lymphocyte protein extraction performed simultaneously with DNA and RNA isolation: application to the study of factors affecting DNA, RNA, and protein recovery from lymphocytes of the oldest individuals.

We describe an optimized procedure for protein extraction performed simultaneously with that of DNA and RNA from a single tissue sample that is, unlike the original protocol, suitable for quantitative studies. This optimized protocol is particularly well adapted to studies where gene regulation at DNA, RNA, and protein levels must be examined simultaneously, and when the amount of starting biological material is limited. We applied this procedure to the study of factors affecting both qualitatively and quantitatively the extraction of DNA, RNA, and proteins from lymphocytes of very old individuals, since we observed variability in the recovery of these molecular species with advanced age. Therefore, we investigated the combined effects of age and time delay between blood collection and lymphocyte isolation on the recovery of DNA, RNA, and proteins simultaneously extracted from Danish nonagenarians and centenarians versus younger adult samples. Our results suggest that neither RNA nor DNA nor protein contents of lymphocytes are altered with aging. However, the quantity of RNA and protein recovery is affected by a 24-h delay in blood processing. This effect is more pronounced in the oldest, particularly for RNA, and may affect data interpretation of age-dependent gene expression studies.

Detection of the peripheral nervous system (PNS)-type glial fibrillary acidic protein (GFAP) and its mRNA in human lymphocytes.

Glial fibrillary acidic protein (GFAP), an astroglial marker, has been detected in the peripheral nervous system (PNS) in a shorter version and its mRNA in a longer form (beta-type) than the brain alpha-type. To determine the characteristics of the GFAP gene expression in nonneural cells, we have investigated its in vivo transcription and translation products in human lymphocytes. Using RT-PCR, we demonstrate that the GFAP gene is transcribed in these cells. Most or all of the mRNA resulting from this transcription was longer than the brain-type at its 5' end and thus may correspond to the beta-type. In addition, immunoblotting of lymphocyte extracts with a monoclonal antibody revealed a 41 KDa fragment instead of the 50 KDa expected from brain GFAP. These results suggest that GFAP expression in lymphocytes is preferentially of the PNS beta-type giving rise to longer mRNA and shorter protein. However, compared to two other astroglial mRNAs (S-100beta and aldolase C) which were synthesized in significant amounts in lymphocytes, GFAP mRNA was detected in minute amounts representing 0.03% of the brain level. This low expression may subserve a special role in lymphocytes since it is translated.

Transcriptional regulation of glial fibrillary acidic protein (GFAP)-mRNA expression during postnatal development of mouse brain.

During mouse brain maturation, GFAP-mRNA undergoes a two-step developmental expression. It increases between birth and day 15 (period of astrocytic proliferation) and then decreases until day 55 (period of astrocytic morphological differentiation). We have developed an in vitro transcription procedure, as a mean to study the part of transcriptional control in this biphasic expression. After RNA synthesis by endogenous RNA polymerases in nuclei isolated from mouse brain (of 3 to 55 days and 217 days), the relative rates of GFAP-mRNA transcripts were analysed by hybridization with a specific cDNA probe. As early as 3 days after birth, the rate of GFAP-mRNA transcripts was maximal, whereas unexpectedly, it showed a significant decrease in mice of 15 days and stayed low until the 55th day. Therefore, a transcriptional control may take place early in mouse brain postnatal development by increasing the transcriptional rate of the GFAP gene in astrocytes, and during the transition from proliferation to differentiation phase of astrocytes (that occurs at the 15th day after birth) by decreasing this rate. However, posttranscriptional events may also occur to modulate the level of the cytoplasmic GFAP-mRNA. In older mice (217 days), the low rate of GFAP-mRNA transcripts found is not concordant with the high cytoplasmic level generally observed in gliosis of the aging brain. Our data suggest posttranscriptional events at this age.

Developmental expression of the glial fibrillary acidic protein mRNA in the central nervous system and in cultured astrocytes.

The expression of glial fibrillary acidic protein (GFAP)-mRNA during mouse brain development and in astroglial primary cultures has been investigated by using two approaches: Northern-blot evaluation using a specific cDNA probe, and cell-free translation associated with immunoprecipitation. During brain maturation (4-56 days postnatal), the GFAP-mRNA underwent a biphasic evolution. An increase was observed between birth and day 15 (i.e., during the period of astroglial proliferation), which was followed by a decrease until day 56 (i.e., during astroglial cell differentiation). At older stages (300 days), an increase was observed, which might reflect gliosis. During astroglial in vitro development (7-32 days in culture), the GFAP-mRNA showed similar variations. An increase, observed during the period of astroglial proliferation (7-18 days), was followed by a decrease which occurred in parallel to marked changes in cell shape, cell process outgrowth, and the organization and accumulation of gliofilaments. During the same culture period (7-32 days), alpha-tubulin mRNA, which was used as an internal standard, did not vary significantly. These results show that the increase of the GFAP protein and of gliofilaments observed both in vivo and in vitro during astroglial differentiation cannot be ascribed to an accumulation of the GFAP-mRNA. It might be that more than one mechanism regulates the levels of free and polymerized GFAP and of its encoding mRNA.