A new multidrug resistance protein at the blood-brain barrier

Porcine brain capillary endothelial cells (PBCEC) cultured in serum-free and hydrocortisone supplemented medium are characterised by high transendothelial electrical resistances and low cell monolayer permeabilities for small solutes very similar to the blood-brain barrier (BBB) in vivo. Differential screening of a subtracted cDNA library disclosed a 2.1-kb mRNA that is overexpressed in hydrocortisone treated PBCEC relative to untreated cells. The mRNA encodes a 656-aa member of the ATP-binding cassette (ABC) superfamily of transporters that we named brain multidrug resistance protein (BMDP). Phylogenetic analysis and multiple sequence alignment showed that porcine BMDP is most related to the human and mouse breast cancer resistance protein (BCRP). Northern blot analysis revealed that BMDP is expressed in brain tissue in vivo and was predominantly localised within the endothelial cells isolated from brain capillaries. Thus, we identified a new transport protein at the BBB that might play an important role in the exclusion of xenobiotics from the brain.     

Isolation of poly(A)+ RNA by paper affinity chromatography

Poly(A)+ RNA was isolated from in vitro short-term-labeled total cytoplasmic RNA of Ehrlich ascites tumor cells by oligo(dT) cellulose chromatography. This poly(A)+ RNA fraction was compared with a poly(A)+ RNA fraction isolated by a new procedure which involves specific binding of poly(A)+ RNA to messenger affinity paper (mAP) and its release in hot (70 degrees C) water. In typical experiments 10-11 micrograms (2.3%) of poly(A)+ RNA can be retained from 500 micrograms of total cytoplasmic RNA per cm2 of mAP in a quick one-step procedure. The poly(A)+ RNA preparations isolated by the two methods proved to be almost identical with respect to their fraction in total cytoplasmic RNA, specific radioactivities, sucrose gradient profiles, and translation assays. Since the isolation of poly(A)+ RNA by mAP is much less time consuming than that by oligo(dT) column chromatography and since the poly(A)+ RNA can be recovered from mAP in small volumes, which avoids further loss during precipitations, it can be advantageously used for preparative isolation of poly(A)+ RNA.     

Biogenesis and cell cycle relationship of poly(A)- actin mRNA in mouse ascites cells

A variety of rapidly growing mammalian cells contain a substantial portion of their actin mRNA in a poly(A)- form. We have used DNA-driven hybridization of a cloned actin cDNA-containing plasmid with pulse-labeled RNA from mouse S-180 ascites cells to examine newly synthesized actin mRNA. Our results indicate that the same proportion of newly synthesized and steady-state actin mRNA (approx. 40%) exists in a poly(A)- deficient form. This suggests that the poly(A)- form arises by some process other than slow cytoplasmic de-adenylation of a poly(A)+ precursor. We have also examined cell cycle-enriched populations of S-180 ascites cells for the presence of poly(A)- actin mRNA. Results from these experiments indicate that cells in G1 phase of the cell cycle contain predominantly poly(A)+ actin mRNA, while the poly(A)- form is restricted to late-S and post-S phase cells.     

Mouse testes contain two size classes of actin mRNA that are differentially expressed during spermatogenesis.

Using several actin isotype-specific cDNA probes, we found actin mRNA of two size classes, 2.1 and 1.5 kilobases (kb), in extracts of polyadenylated and nonpolyadenylated RNA from sexually mature CD-1 mouse testes. Although the 2.1-kb sequence was present in both meiotic and postmeiotic testicular cell types, it decreased manyfold in late haploid cells. The 1.5-kb actin sequence was not detectable in meiotic pachytene spermatocytes (or in liver or kidney cells), but was present in round and elongating spermatids and residual bodies. To differentiate between the beta- and gamma-actin mRNAs, we isolated a cDNA, pMGA, containing the 3' untranslated region of a mouse cytoplasmic actin that has homology to the 3' untranslated region of a human gamma-actin cDNA but not to the 3' untranslated regions of human alpha-, beta-, or cardiac actins. Dot blot hybridizations with pMGA detected high levels of presumptive gamma-actin mRNA in pachytene spermatocytes and round spermatids, with lower amounts found in elongating spermatids. Hybridization with the 3' untranslated region of a rat beta-actin probe revealed that round spermatids contained higher levels of beta-actin mRNA than did pachytene spermatocytes or residual bodies. Both probes hybridized to the 2.1-kb actin mRNA but failed to hybridize to the 1.5-kb mRNA.     

Identification and developmental expression of a smooth-muscle gamma-actin in postmeiotic male germ cells of mice.

Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.     

Direct isolation of poly(A)+ RNA from 4 M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture

LNA oligonucleotides constitute a class of bicyclic RNA analogues having an exceptionally high affinity for their complementary DNA and RNA target molecules. We here report a novel method for highly efficient isolation of intact poly(A)+ RNA using an LNA-substituted oligo(dT) affinity ligand, based on the increased affinity of LNA-T for complementary poly(A) tracts. Poly(A)+ RNA was isolated directly from 4 M guanidine thiocyanate-lysed Caenorhabditis elegans worm extracts as well as from lysed human K562 and vincristine-resistant K562/VCR leukemia cells using LNA_2.T oligonucleotide as an affinity probe, in which every second thymidine was substituted by LNA thymidine. In accordance with the significantly increased stability of the LNA_2.T-A duplexes in 4 M GuSCN, we obtained a 30- to 50-fold mRNA yield increase using the LNA-substituted oligo(T) affinity probe compared with DNA-oligo(dT)-selected mRNA samples. The LNA_2.T affinity probe was, furthermore, highly efficient in isolation of poly(A)+ RNA in a low salt concentration range of 50-100 mM NaCl in poly(A) binding buffer, as validated by selecting the mRNA pools from total RNA samples extracted from different Saccharomyces cerevisiae strains, followed by northern blot analysis. Finally, we demonstrated the utility of the LNA-oligo(T)-selected mRNA in quantitative real-time PCR by analysing the relative expression levels of the human mdr1 multidrug resistance gene in the two K562 cell lines employing pre-validated Taqman assays. Successful use of the NH2-modified LNA_2.T probe in isolation of human mRNA implies that the LNA-oligo(T) method could be automated for streamlined, high throughput expression profiling by real-time PCR by covalently coupling the LNA affinity probe to solid, pre-activated surfaces, such as microtiter plate wells or magnetic particles.     

Analysis of CD4 gene expression in human fetal brain and neuroblasts

1. The expression of the gene codifying for CD4, the most important human immunodeficiency virus type 1 (HIV-1) receptor molecule, was analyzed in 11 fetal brains at various gestational ages and in 9 human neuroblastoma (NB) cell lines. CD4 gene expression in fetal and malignant neural cells was then compared with that observed in a hematopoietic cell line and adult hippocampus. 2. In addition, CD4 mRNA was evaluated in two NB cell lines induced to differentiate in vitro with retinoic acid (RA) or 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine (H7), a protein kinase C inhibitor. 3. All fetal brains and NB cell lines express a 1.8-kb signal when hybridized with pT4BcDNA probe, while a 3.0-kb signal such as observed in hematopoietic human cells was found in 1 of 11 fetal brains and in 0 of 9 NB cell lines. The 1.8-kb signal was lost in all analyzed poly(A)+ mRNA samples. 4. Moreover, CD4 gene expression was not induced in either RA- or H7-treated NB cells at any tested time and dose. The analysis of NB cells by polymerase chain reaction failed to demonstrate CD4 expression in either poly(A)+ or poly(A)- RNA. 5. In conclusion, the results show that the 1.8-kb signal observed in RNA extracted from fetal or transformed human neural cells is probably due to an aspecific hybridization. However, the gene codifying for CD4 can rarely be expressed by fetal brain cells early during gestation, in still unclear circumstances.