Increased expression of importin-ß, exportin-5 and nuclear transportable proteins in Alzheimer's disease aids anatomic pathologists in its diagnosis.

Understanding the metabolic profile of neurons with the hyperphosphorylated tau protein characteristic of Alzheimer's disease is essential to unraveling new potential therapies and diagnostics for the surgical pathologist. We stratified 75 brain tissues from Alzheimer's disease into hyperphosphorylated tau positive or negative and did co-expression analyses and qRTPCR for importin-ß and exportin-5 plus several bcl2 family members and compared the data to controls, Down's dementia and Parkinson's disease. There was a significant increase in the expression of importin-ß and exportin-5 in Alzheimer's disease relative to the three other categories (each p value<0.0001) where each protein co-localized with hyperphosphorylated tau. Both apoptotic and anti-apoptotic proteins were each significantly increased in Alzheimer's disease relative to the three other groups. Neurons with hyperphosphorylated tau in Alzheimer's disease have the profile of metabolically active cells including increased exportin-5 and importin-ß mRNA and proteins which indicates that immunohistochemistry testing of these proteins may aid the surgical pathologist in making a definitive diagnosis.

Cell-specific RNA aptamer against human CCR5 specifically targets HIV-1 susceptible cells and inhibits HIV-1 infectivity.

The C-C chemokine receptor type 5 (CCR5) is a receptor expressed by T cells and macrophages that serves as a coreceptor for macrophage-tropic HIV-1. Loss of CCR5 is associated with resistance to HIV-1. Here, we combine the live-cell-based SELEX with high-throughput sequencing technology to generate CCR5 RNA aptamers capable of specifically targeting HIV-1 susceptible cells (as small interfering RNA [siRNA] delivery agent) and inhibiting HIV-1 infectivity (as antiviral agent) via block of the CCR5 required for HIV-1 to enter cells. One of the best candidates, G-3, efficiently bound and was internalized into human CCR5-expressing cells. The G-3 specifically neutralized R5 virus infection in primary peripheral blood mononuclear cells, and in vivo generated human CD4(+) T cells with a nanomolar inhibitory concentration 50%. G-3 was also capable of transferring functional siRNAs to CCR5-expressing cells. Collectively, the cell-specific, internalizing, CCR5-targeted aptamers and aptamer-siRNA conjugates offer promise for overcoming some of the current challenges of drug resistance in HIV-1 by providing cell-type- or tissue-specific delivery of various therapeutic moieties.

Limb-girdle muscular dystrophy 1F is caused by a microdeletion in the transportin 3 gene.

In 2001, we reported linkage of an autosomal dominant form of limb-girdle muscular dystrophy, limb-girdle muscular dystrophy 1F, to chromosome 7q32.1-32.2, but the identity of the mutant gene was elusive. Here, using a whole genome sequencing strategy, we identified the causative mutation of limb-girdle muscular dystrophy 1F, a heterozygous single nucleotide deletion (c.2771del) in the termination codon of transportin 3 (TNPO3). This gene is situated within the chromosomal region linked to the disease and encodes a nuclear membrane protein belonging to the importin beta family. TNPO3 transports serine/arginine-rich proteins into the nucleus, and has been identified as a key factor in the HIV-import process into the nucleus. The mutation is predicted to generate a 15-amino acid extension of the C-terminus of the protein, segregates with the clinical phenotype, and is absent in genomic sequence databases and a set of >200 control alleles. In skeletal muscle of affected individuals, expression of the mutant messenger RNA and histological abnormalities of nuclei and TNPO3 indicate altered TNPO3 function. Our results demonstrate that the TNPO3 mutation is the cause of limb-girdle muscular dystrophy 1F, expand our knowledge of the molecular basis of muscular dystrophies and bolster the importance of defects of nuclear envelope proteins as causes of inherited myopathies.

Distinguishing Barrett gastric foveolar dysplasia from reactive cardiac mucosa in gastroesophageal reflux disease.

Morphologic dysplasia remains the criterion standard of cancer risk in Barrett esophagus but poses many challenges including distinction from reactive inflammatory change. Gastric foveolar dysplasia, a newly described subtype comprising 15% to 20% of Barrett dysplasia, overlaps with reactive cardiac mucosa in gastroesophageal reflux disease (GERD). Despite the clinical importance of accurate distinction, the issue has not been studied. Review of 3698 biopsies from 461 Barrett patients yielded 160 biopsies with Barrett gastric foveolar dysplasia (74 low grade and 86 high grade). These were compared with inflamed cardia from 80 patients with GERD. Immunohistochemistry was performed for Lgl2, MUC2, MUC5AC, and MUC6. Comparing GERD with Barrett gastric foveolar dysplasia, surface nuclear stratification (85% versus 0%, P < .00001), upper mucosa-limited atypia (80% versus 0%, P < .0001), villiform architecture (52% versus 4%; P < .0001), full-thickness mucosal atypia (0% versus 100%, P < .00001), and crowded glandular architecture (0% versus 75%, P < .00001) all proved useful. Cytologic features were less helpful. Comparing low-grade gastric dysplasia alone, because its distinction from reactive cardia may be even more challenging, the listed features all remained significant. Loss or aberrant Lgl2 expression was much more typical of dysplasia (12% versus 99%; P = .0001). MUC proteins did not distinguish the groups. Surface nuclear stratification, "top-heavy" atypia, and noncrowded, villiform architecture were highly characteristic of reactive cardiac atypia in GERD, in comparison with the monolayered nuclei in crowded glands occupying the full mucosal thickness in Barrett gastric foveolar dysplasia. Loss or aberrant Lgl2 staining was useful in identifying Barrett gastric foveolar dysplasia.

Expression of the nuclear transport protein importin ß-1 and its association with the neurokinin 3 receptor in the rat hypothalamus following acute hyperosmotic challenge.

The tachykinin NK3 receptor (NK3R) is a G-protein coupled receptor that is activated, internalized, and trafficked to the nuclei of magnocellular neurons in the paraventricular nucleus of the hypothalamus (PVN) in response to acute hyperosmolarity. The lack of information on the nuclear import pathway raises concerns about the physiological role of nuclear NK3R. NK3R contains a nuclear localizing sequence (NLS) and this raises the possibility that importins are involved in transport of NK3R through the nuclear pore complex. The following experiments utilized: (1) co-immunoprecipitation to determine if NK3R is associated with importin ß-1 following activation in response to acute hyperosmolarity in vivo, and (2) immuno-neutralization of importin ß-1 in vitro to determine if nuclear transport of NK3R was blocked. Rats were given an i.v. injection of hypertonic saline (2 M) and 10 min after the infusion, the PVN was removed and homogenized. Importin ß-1 co-immunoprecipitated with the NK3R following treatment with 2 M NaCl, but not following isotonic saline treatment. Immuno-neutralization of importin ß-1 decreased the transport of NK3R into the nuclei in a time dependent fashion. The results indicate that in response to acute hyperosmotic challenge, NK3R associates with importin ß-1 which enables the nuclear transport of NK3R. This is the first in vivo study linking importin ß-1 and the nuclear transport of a G protein coupled receptor, the NK3R, in brain.

The Karyopherin proteins, Crm1 and Karyopherin beta1, are overexpressed in cervical cancer and are critical for cancer cell survival and proliferation.

The Karyopherin proteins are involved in nucleo-cytoplasmic trafficking and are critical for protein and RNA subcellular localization. Recent studies suggest they are important in nuclear envelope component assembly, mitosis and replication. Since these are all critical cellular functions, alterations in the expression of the Karyopherins may have an impact on the biology of cancer cells. In this study, we examined the expression of the Karyopherins, Crm1, Karyopherin beta1 (Kpnbeta1) and Karyopherin alpha2 (Kpnalpha2), in cervical tissue and cell lines. The functional significance of these proteins to cancer cells was investigated using individual siRNAs to inhibit their expression. Microarrays, quantitative RT-PCR and immunofluorescence revealed significantly higher expression of Crm1, Kpnbeta1 and Kpnalpha2 in cervical cancer compared to normal tissue. Expression levels were similarly elevated in cervical cancer cell lines compared to normal cells, and in transformed epithelial and fibroblast cells. Inhibition of Crm1 and Kpnbeta1 in cancer cells significantly reduced cell proliferation, while Kpnalpha2 inhibition had no effect. Noncancer cells were unaffected by the inhibition of Crm1 and Kpnbeta1. The reduction in proliferation of cancer cells was associated with an increase in a subG1 population by cell cycle analysis and Caspase-3/7 assays revealed increased apoptosis. Crm1 and Kpnbeta1 siRNA-induced apoptosis was accompanied by an increase in the levels of growth inhibitory proteins, p53, p27, p21 and p18. Our results demonstrate that Crm1, Kpnbeta1 and Kpnalpha2 are overexpressed in cervical cancer and that inhibiting the expression of Crm1 and Kpnbeta1, not Kpnalpha2, induces cancer cell death, making Crm1 and Kpnbeta1 promising candidates as both biomarkers and potential anticancer therapeutic targets.

Expression of nuclear transport importins beta 1 and beta 3 is regulated during rodent spermatogenesis.

Spermatogenic differentiation requires progressive gene expression changes, and proteins required for this must be transported into the nucleus. Many of these contain a nuclear localization signal and are likely to be transported by importin protein family members, each of which recognizes and transports distinct cargo proteins. We hypothesized that importins, as modulators of protein nuclear access, would display distinct expression profiles during spermatogenesis, indicating their potential to regulate key steps in cellular differentiation. This was tested throughout testicular development in rodents. Real-time PCR analysis of postnatal mouse testes revealed changing expression levels of Knpb1 (encoding importin beta 1) and Ranbp5 (encoding beta 3) mRNAs, with Knpb1 highest at 26 days postpartum and Ranbp5 highest in Day 26 and adult testis. Their distinctive cellular expression patterns visualized using in situ hybridization and immunohistochemistry were identical in mouse and rat testes where examined. Within the seminiferous epithelium, Knpb1 mRNA and importin beta1 protein were detected within mitotic Sertoli and germ cells during fetal and early postnatal development, becoming restricted to spermatogonia and spermatocytes in adulthood. Importin beta 3 protein in fetal germ cells displayed a striking difference in intracellular localization between male and female gonads. In adult testes, Ranbp5 mRNA was detected in round spermatids and importin beta 3 protein in elongating spermatids. This is the first comprehensive in situ demonstration of developmentally regulated synthesis of nuclear transport components. The contrasting expression patterns of importins beta 1 and 3 identify them as candidates for regulating nuclear access of factors required for developmental switches.

Enhanced nuclear import and transfection efficiency of plasmid DNA using streptavidin-fused importin-beta.

In order to enhance the nuclear import of exogenous genes, novel plasmid DNA/importin-beta conjugates, which consist of a biotinylated plasmid DNA and a recombinant streptavidin-fused importin-beta, were prepared. The spacer length between plasmid DNA and biotin and the number of introduced biotin were adjusted. The microinjection of plasmid DNA/importin-beta conjugates into the cytoplasm of NIH3T3 cells resulted in the nuclear localization of conjugates and the higher expression efficiency, compared to intact plasmid DNA alone. These results indicate that plasmid DNA/importin-beta conjugates would be an important tool to enhance the nuclear localization of exogenous DNA in non-viral gene delivery system.

Crystallization and preliminary crystallographic analysis of the importin-beta-SREBP-2 complex.

The nuclear-transport protein importin-beta mediates the nuclear import of the transcription factor SREBP-2 without requiring adaptor proteins such as importin-alpha. An importin-beta-SREBP-2 HLHZ domain complex was purified and crystallized. The crystals belong to space group P2(1)2(1)2(1) and show diffraction to at least 3.0 A resolution. The unit-cell parameters are a = 101.0, b = 113.2, c = 240.0 A. Structure determination using the MAD or SAD method is under way.

C-terminal kinesin motor KIFC1 participates in acrosome biogenesis and vesicle transport.

We have identified a possible role for the KIFC1 motor protein in formation of the acrosome, an organelle unique to spermatogenesis. KIFC1, a C-terminal kinesin motor, first appears on membrane-bounded organelles (MBOs) in the medulla of early spermatids followed by localization to the acrosomal vesicle. KIFC1 continues to be present on the acrosome of elongating spermatids as it flattens on the spermatid nucleus; however, increasing amounts of KIFC1 are found at the caudal aspect of the spermatid head and in distal cytoplasm. The KIFC1 motor is also found in the nucleus of very immature round spermatids just prior to its appearance on the acrosome. In some cases, KIFC1 appears localized just below the nuclear membrane adjacent to the subacrosomal membrane. We demonstrate that KIFC1 is associated with importin beta and colocalizes with this nuclear transport factor on curvilinear structures associated with the spermatid nuclei. These data support a model in which KIFC1, perhaps in association with nuclear factors, assists in the formation and/or elongation of the spermatid acrosome. This article represents the first demonstration of a direct association of a molecular motor with the spermatid acrosome, the formation of which is essential for fertilization.

Ran GTPase cycle and importins alpha and beta are essential for spindle formation and nuclear envelope assembly in living Caenorhabditis elegans embryos.

The small GTPase Ran has been found to play pivotal roles in several aspects of cell function. We have investigated the role of the Ran GTPase cycle in spindle formation and nuclear envelope assembly in dividing Caenorhabditis elegans embryos in real time. We found that Ran and its cofactors RanBP2, RanGAP, and RCC1 are all essential for reformation of the nuclear envelope after cell division. Reducing the expression of any of these components of the Ran GTPase cycle by RNAi leads to strong extranuclear clustering of integral nuclear envelope proteins and nucleoporins. Ran, RanBP2, and RanGAP are also required for building a mitotic spindle, whereas astral microtubules are normal in the absence of these proteins. RCC1(RNAi) embryos have similar abnormalities in the initial phase of spindle formation but eventually recover to form a bipolar spindle. Irregular chromatin structures and chromatin bridges due to spindle failure were frequently observed in embryos where the Ran cycle was perturbed. In addition, connection between the centrosomes and the male pronucleus, and thus centrosome positioning, depends upon the Ran cycle components. Finally, we have demonstrated that both IMA-2 and IMB-1, the homologues of vertebrate importin alpha and beta, are essential for both spindle assembly and nuclear formation in early embryos.