Emphysematous pyelonephritis.
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Acute Kidney Injury↗
Biomedical subjects
J Reiser
Publications and source records attributed to J Reiser.
Retrovirus molecular conjugates: a versatile and efficient gene transfer vector system for primitive human hematopoietic progenitor cells.
In principle, transient nongenetic modification of a noninfectious gene transfer virus enabling a one time infection and transduction of human cells could eliminate the risk of formation of replication competent virus. Formation of a molecular conjugate vector by conjugation of noninfective ecotropic murine Moloney leukemia virus to polylysine (eMMLV-PL) enabled high-efficiency transduction of human HPC using in vitro and in vivo assays. Xenotransplanted NOD-SCID mice durably expressed the transgene in human leukocytes and human progenitor cells with eMMLV-PL achieving three-fold increased transduction efficiency when directly compared to optimized amphotropic MMLV (aMMLV) transduction. Both aMMLV and eMMLV assembled conjugate vectors showed similar transduction efficiency indicating predominant polylysine-mediated uptake. Integration of retroviral sequences was determined from individual human HPC recovered from eMMLV-PL-xenotransplanted animals. This simple and versatile concept of conjugate gene transfer vectors has the potential to enhance transduction efficiency as well as to improve certain safety aspects of human gene therapy. Moreover, because it permits effective cellular internalization of particles, this concept of molecular conjugates can be used as research tool to investigate the interactions of otherwise noninfectious viruses or modified viral particles at the genomic level.
Animals↗
Process formation of podocytes: morphogenetic activity of microtubules and regulation by protein serine/threonine phosphatase PP2A.
Podocytes possess major processes containing microtubules (MTs) and intermediate filaments and foot processes containing actin filaments (AFs) as core cytoskeletal elements. Although the importance of these cytoskeletal elements for maintaining podocyte processes was previously shown, so far no data are available concerning the developmental regulation of podocyte process formation. A conditionally immortalized mouse podocyte cell line, which can be induced to develop processes similar to those found in vivo, was treated with various reagents to disrupt cytoskeletal elements or to inhibit protein phosphatases. MTs colocalized with vimentin intermediate filaments but not with AFs. After AF disassembly, major processes were maintained, whereas after depolymerization of MTs, podocytes lost their processes, rounded up, and maintained only actin-based peripheral projections. Suppression of MT elongation by nanomolar vinblastine or inhibition of serine/threonine phosphatase PP2A with okadaic acid abolished process formation. PP2A was expressed in undifferentiated but not in differentiated podocytes. One- and two-dimensional western blot analyses revealed a dose-dependent increase in serine/threonine phosphorylation after okadaic acid treatment. Hence, morphogenetic activity of MTs induces podocyte process formation via serine/threonine protein dephosphorylation by PP2A. These results may open new avenues for understanding the signaling mechanism underlying podocyte cytoskeleton alterations during development and in glomerular diseases.
Actin Cytoskeleton↗
Involvement of lipid rafts in nephrin phosphorylation and organization of the glomerular slit diaphragm.
NPHS1 has recently been identified as the gene whose mutations cause congenital nephrotic syndrome of the Finnish type. The respective gene product nephrin is a transmembrane protein expressed in glomerular podocytes and primarily localized to the glomerular slit diaphragm. This interpodocyte junction functions in the glomerular filtration by restricting the passage of plasma proteins into the urinary space in a size-selective manner. The functional role of nephrin in this filtration process is so far not very well understood. In this study, we show that nephrin associates in an oligomerized form with signaling microdomains, also known as lipid rafts, and that these localize to the slit diaphragm. We also show that the nephrin-containing rafts can be immunoisolated with the 27A antibody recognizing a podocyte-specific 9-O-acetylated GD3 ganglioside. In a previous study it has been shown that the in vivo injection of this antibody leads to morphological changes of the filtration slits resembling foot process effacement. Here, we report that, in this model of foot process effacement, nephrin dislocates to the apical pole of the narrowed filtration slits and also that it is tyrosine phosphorylated. We suggest that lipid rafts are important in the spatial organization of the glomerular slit diaphragm under physiological and pathological conditions.
Animals↗
Survival effects of pigment epithelium-derived factor expressed by a lentiviral vector in rat cerebellar granule cells.
We have previously shown that pigment epithelium-derived factor (PEDF) acts as a survival factor for cerebellar granule cells (CGCs), by blocking apoptotic death, and can also protect these cells against glutamate-induced neurotoxicity. In preparation for gene therapy studies, pseudotyped HIV-1-based lentiviral vectors containing the PEDF gene, as well as either green fluorescent protein or beta-galactosidase, were prepared. These bicistronic vectors are unique in that they express two genes efficiently under one promoter. Primary cell cultures of CGCs from postnatal day 8 rats were infected with the vectors encoding PEDF. RT-PCR demonstrated expression of mRNA and Western blot analysis confirmed that infected CGCs secrete PEDF protein to the medium. Assays for cell survival demonstrated that PEDF-infected cells were significantly more protected compared with mock-infected controls for 6-8 days in culture, as well as against induced apoptosis. The PEDF vectors expressing tat (trans-acting transcription factor) provided more protection than the tat(-) vectors. These results demonstrate that while the lentiviral vectors expressing PEDF are as neuroprotective as the protein itself for CGCs, the vectors have the advantage of providing long-lasting expression of PEDF protein, which will be more effective in in vivo studies. The present results suggest that this system may be useful for gene therapy for neurodegenerative disorders.
Animals↗
Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin.
NPHS2 was recently identified as a gene whose mutations cause autosomal recessive steroid-resistant nephrotic syndrome. Its product, podocin, is a new member of the stomatin family, which consists of hairpin-like integral membrane proteins with intracellular NH(2)- and COOH-termini. Podocin is expressed in glomerular podocytes, but its subcellular distribution and interaction with other proteins are unknown. Here we show, by immunoelectron microscopy, that podocin localizes to the podocyte foot process membrane, at the insertion site of the slit diaphragm. Podocin accumulates in an oligomeric form in lipid rafts of the slit diaphragm. Moreover, GST pull-down experiments reveal that podocin associates via its COOH-terminal domain with CD2AP, a cytoplasmic binding partner of nephrin, and with nephrin itself. That podocin interacts with CD2AP and nephrin in vivo is shown by coimmunoprecipitation of these proteins from glomerular extracts. Furthermore, in vitro studies reveal direct interaction of podocin and CD2AP. Hence, as with the erythrocyte lipid raft protein stomatin, podocin is present in high-order oligomers and may serve a scaffolding function. We postulate that podocin serves in the structural organization of the slit diaphragm and the regulation of its filtration function.
Adaptor Proteins, Signal Transducing↗
Podocyte biology: a footstep further.
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Animals↗
Intercellular delivery of a herpes simplex virus VP22 fusion protein from cells infected with lentiviral vectors.
Effective gene therapy depends on the efficient transfer of therapeutic genes and their protein products to target cells. Lentiviral vectors appear promising for virus-mediated gene delivery and long-term expression in nondividing cells. The herpes simplex virus type 1 tegument protein VP22 has recently been shown to mediate intercellular transport of proteins, raising the possibility that it may be helpful in a setting where the global delivery of therapeutic proteins is desired. To investigate the effectiveness of lentiviral vectors to deliver genes encoding proteins fused to VP22, and to test whether the system is sufficiently potent to allow protein delivery from transduced cells in vitro and in vivo, fusion constructs of VP22 and the enhanced green fluorescent protein (EGFP) were prepared and delivered into target cells by using HIV-1-based lentiviral vectors. To follow the spread of VP22-EGFP to other cells, transduced COS-7 cells were coplated with a number of different cell types, including brain choroid plexus cells, human endothelial cells, H9 cells, and HeLa cells. We found that VP22-EGFP fusion proteins were transported from transduced cells to recipient cells and that such fusion proteins accumulated in the nucleus and in the cytoplasm of such cells. To determine the ability to deliver fusion proteins in vivo, we injected transduced H9 cells as well as the viral vector directly into the brain of mice. We present evidence that VP22-EGFP fusion proteins were transported effectively from lentivirus transduced cells in vivo. We also show that the VP22-EGFP fusion protein encoded by the lentivirus is transported between cells. Our data indicate that such fusion proteins are present in the nucleus and in the cytoplasm of neighboring cells. Therefore, lentiviral vectors may provide a potent biological system for delivering genes encoding therapeutic proteins fused to VP22.
Animals↗
Modified human immunodeficiency virus-based lentiviral vectors display decreased sensitivity to trans-dominant Rev.
As a first step toward the development of HIV-based conditionally replicating defective interfering particles expressing trans-dominant Rev (TdRev), we studied whether mutation of the splicing signals and replacement of the RRE by the SRV-1 CTE would render these vectors less sensitive to TdRev. Vectors with mutations in the splicing signals (SD-/RRE+) yielded high titers (5 X 10(6) CFU/ml) and showed higher levels of cytoplasmic unspliced mRNA than the corresponding SD+/RRE+ vectors either in the absence of Rev, in the presence of TdRev, or in the presence of both TdRev and Rev. Proviral copies of SD-/RRE+ vectors were rescued more efficiently than SD+/RRE+ vectors when TdRev was expressed. Vectors with the SRV-1 CTE (SD+/CTE+ and SD-/CTE+) expressed high levels of cytoplasmic unspliced mRNA in the absence of Rev expression. Titers obtained with the SD-/CTE+ vectors (10(6) CFU/ml) were higher than the titers obtained with SD+/CTE+ vectors. We also tested the effect of other structural modifications such as the orientation of the expression cassette and the presence of the central polypurine tract (cPPT/CTS). We show that an expression cassette cloned in the reverse orientation with respect to the LTRs or elimination of the cPPT/CTS element severely affected vector titers. We also demonstrated that these vectors can be efficiently mobilized from their proviral state by HIV trans-complementing functions, and transduced into secondary target cells without suffering any genomic rearrangement.
Base Sequence↗
Production and concentration of pseudotyped HIV-1-based gene transfer vectors.
Strategies to generate highly concentrated HIV-1 vector pseudotypes involving different envelope (Env) proteins including the vesicular stomatitis virus (VSV) G glycoprotein, the Moloney murine leukemia virus (MLV) 4070A amphotropic Env and the rabies G glycoprotein were established. Virus stocks were prepared by transient transfection using standard cell culture media or serum-free media. Such stocks were concentrated 50- to 300-fold by ultracentrifugation or by ultrafiltration using Centricon Plus-80 units yielding titers of up to 109transducing units per milliliter. There was no loss in titer with any of the pseudotypes tested. Thus, like lentiviral vectors pseudotyped with VSV-G, HIV-1-based vectors pseudotyped with the MLV 4070A amphotropic Env and the rabies G glycoprotein resist inactivation during concentration. This opens up the possibility to generate highly concentrated HIV-1 vector stocks carrying alternative Env proteins on a large scale.
Cell Line↗
Regulation of mouse podocyte process dynamics by protein tyrosine phosphatases rapid communication.
BACKGROUND: Effacement of podocyte foot processes occurs early in many glomerular diseases associated with proteinuria and is accompanied by a reorganization of the actin cytoskeleton. The molecular mechanisms regulating these structural changes are poorly understood. METHODS: To address these questions, we analyzed the effect of the polycation, protamine sulfate (PS), and puromycin aminonucleoside (PA) on the morphology, cytoskeleton, and tyrosine phosphorylation of differentiated process-bearing cultured podocytes. RESULTS: PS and PA induced similar profound morphological alterations, including retraction and detachment of podocyte processes from the extracellular matrix (ECM). The effects of PS occurred within six hours, whereas PA showed its most severe effects after 72 hours. Structural changes included reorganization of the actin cytoskeleton and focal contacts and were accompanied by an increase in tyrosine phosphorylation. The same effects were induced by application of vanadate, an inhibitor of protein tyrosine phosphatases (PTPs), suggesting that PTPs regulate podocyte process structure. Since disruption of the actin cytoskeleton with cytochalasin B protected the cells from PS-induced effacement and detachment, cytoplasmic PTPs were implicated in these events. Using reverse transcription-polymerase chain reaction (RT-PCR), we demonstrated the expression of four cytoplasmic PTPs in podocytes: SHP-2, PTP-PEST, PTP-1B, and PTP-36. CONCLUSIONS: These studies indicate an important role for cytoplasmic PTPs as regulators of podocyte process dynamics. Future studies will aim at restoring the normal foot process architecture of podocytes in glomerular diseases associated with proteinuria by modulating the activity of cytoplasmic PTPs.
Actins↗
Development of multigene and regulated lentivirus vectors.
Previously we described safe and efficient three-component human immunodeficiency virus type 1 (HIV-1)-based gene transfer systems for delivery of genes into nondividing cells (H. Mochizuki, J. P. Schwartz, K. Tanaka, R. O. Brady, and J. Reiser, J. Virol. 72:8873-8883, 1998). To apply such vectors in anti-HIV gene therapy strategies and to express multiple proteins in single target cells, we have engineered HIV-1 vectors for the concurrent expression of multiple transgenes. Single-gene vectors, bicistronic vectors, and multigene vectors expressing up to three exogenous genes under the control of two or three different transcriptional units, placed within the viral gag-pol coding region and/or the viral nef and env genes, were designed. The genes encoding the enhanced version of green fluorescent protein (EGFP), mouse heat-stable antigen (HSA), and bacterial neomycin phosphotransferase were used as models whose expression was detected by fluorescence-activated cell sorting, fluorescence microscopy, and G418 selection. Coexpression of these reporter genes in contact-inhibited primary human skin fibroblasts (HSFs) persisted for at least 6 weeks in culture. Coexpression of the HSA and EGFP reporter genes was also achieved following cotransduction of target cells using two separate lentivirus vectors encoding HSA and EGFP, respectively. For the regulated expression of transgenes, tetracycline (Tet)-regulatable lentivirus vectors encoding the reverse Tet transactivator (rtTA) and EGFP controlled by a Tet-responsive element (TRE) were constructed. A binary HIV-1-based vector system consisting of a lentivirus encoding rtTA and a second lentivirus harboring a TRE driving the EGFP reporter gene was also designed. Doxycycline-modulated expression of the EGFP transgene was confirmed in transduced primary HSFs. These versatile vectors can potentially be used in a wide range of gene therapy applications.
Animals↗
Child maltreatment, other trauma exposure, and posttraumatic symptomatology among children with oppositional defiant and attention deficit hyperactivity disorders.
Consecutive child psychiatric outpatient admissions with disruptive behavior or adjustment disorders were assessed by validated instruments for trauma exposure and posttraumatic stress disorder (PTSD) symptoms and other psychopathology. Four reliably diagnosed groups were defined in a retrospective case-control design: Attention Deficit Hyperactivity Disorder (ADHD), Oppositional Defiant Disorder (ODD), comorbid ADHD-ODD, and adjustment disorder controls. ODD and (although to a lesser extent) ADHD were associated with a history of physical or sexual maltreatment. PTSD symptoms were most severe if (a) ADHD and maltreatment co-occurred or (b) ODD and accident/illness trauma co-occurred. The association between ODD and PTSD Criterion D (hyperarousal/hypervigilance) symptoms remained after controlling for overlapping symptoms, but the association of ADHD with PTSD symptoms was largely due to an overlapping symptom. These findings suggest that screening for maltreatment, other trauma, and PTSD symptoms may enhance prevention, treatment, and research concerning childhood disruptive behavior disorders.
Adjustment Disorders↗
Directed membrane transport is involved in process formation in cultured podocytes.
Mature glomerular visceral epithelial cells, or podocytes, are unique cells with a complex cell architecture. Characteristically, they possess a highly branched array of major processes and foot processes, which are essential for glomerular filtration in the kidney. A podocyte cell line with the potential to exhibit many features of differentiated podocytes, particularly the formation of cell processes, was recently established. In this study, it is shown that directed membrane transport is involved in process formation in cultured podocytes. The well-characterized vesicular stomatitis virus G was used as a marker protein for the biosynthetic pathway in these cells. It seems that newly synthesized vesicular stomatitis virus G is preferentially delivered into the cell processes of the podocytes, where it is colocalized with known regulators of vesicular transport from the Golgi apparatus to the plasma membrane, such as the small GTPase rab8 and the sec6/sec8 complex. To determine the role of vesicular transport in process formation, cells were treated with brefeldin A, a drug that disrupts the trafficking of post-Golgi transport vesicles. As a result, the podocytes reversibly lost their ability to form processes. These findings suggest that podocytes are dependent on a constant fresh source of lipids and proteins to form their processes.
Animals↗
Nonuniform microtubular polarity established by CHO1/MKLP1 motor protein is necessary for process formation of podocytes.
Podocytes are unique cells that are decisively involved in glomerular filtration. They are equipped with a complex process system consisting of major processes and foot processes whose function is insufficiently understood (Mundel, P., and W. Kriz. 1995. Anat. Embryol. 192:385-397). The major processes of podocytes contain a microtubular cytoskeleton. Taking advantage of a recently established cell culture system for podocytes with preserved ability to form processes (Mundel, P., J. Reiser, A. Zúñiga Mejía Borja, H. Pavenstädt, G.R. Davidson, W. Kriz, and R. Zeller. 1997b. Exp. Cell Res. 36:248-258), we studied the functional significance of the microtubular system in major processes. The following data were obtained: (a) Microtubules (MTs) in podocytes show a nonuniform polarity as revealed by hook-decoration. (b) CHO1/ MKLP1, a kinesin-like motor protein, is associated with MTs in podocytes. (c) Treatment of differentiating podocytes with CHO1/MKLP1 antisense oligonucleotides abolished the formation of processes and the nonuniform polarity of MTs. (d) During the recovery from taxol treatment, taxol-stabilized (nocodazole- resistant) MT fragments were distributed in the cell periphery along newly assembled nocodazole-sensitive MTs. A similar distribution pattern of CHO1/MKLP1 was found under these circumstances, indicating its association with MTs. (e) In the recovery phase after complete depolymerization, MTs reassembled exclusively at centrosomes. Taken together, these findings lead to the conclusion that the nonuniform MT polarity in podocytes established by CHO1/MKLP1 is necessary for process formation.
Animals↗
High-titer human immunodeficiency virus type 1-based vector systems for gene delivery into nondividing cells.
Previously we designed novel pseudotyped high-titer replication defective human immunodeficiency virus type 1 (HIV-1) vectors to deliver genes into nondividing cells (J. Reiser, G. Harmison, S. Kluepfel-Stahl, R. O. Brady, S. Karlsson, and M. Schubert, Proc. Natl. Acad. Sci. USA 93:15266-15271, 1996). Since then we have made several improvements with respect to the safety, flexibility, and efficiency of the vector system. A three-plasmid expression system is used to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells with a defective packaging construct, a plasmid coding for a heterologous envelope (Env) protein, and a vector construct harboring a reporter gene such as neo, ShlacZ (encoding a phleomycin resistance/beta-galactosidase fusion protein), HSA (encoding mouse heat-stable antigen), or EGFP (encoding enhanced green fluorescent protein). The packaging constructs lack functional Vif, Vpr, and Vpu proteins and/or a large portion of the Env coding region as well as the 5' and 3' long terminal repeats, the Nef function, and the presumed packaging signal. Using G418 selection, we routinely obtained vector particles pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) with titers of up to 8 x 10(7) CFU/microgram of p24, provided that a functional Tat coding region was present in the vector. Vector constructs lacking a functional Tat protein yielded titers of around 4 x 10(6) to 8 x 10(6) CFU/microgram of p24. Packaging constructs with a mutation within the integrase (IN) core domain profoundly affected colony formation and expression of the reporter genes, indicating that a functional IN protein is required for efficient transduction. We explored the abilities of other Env proteins to allow formation of pseudotyped HIV-1 particles. The rabies virus and Mokola virus G proteins yielded high-titer infectious pseudotypes, while the human foamy virus Env protein did not. Using the improved vector system, we successfully transduced contact-inhibited primary human skin fibroblasts and postmitotic rat cerebellar neurons and cardiac myocytes, a process not affected by the lack of the accessory proteins.
Animals↗
Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.
Mature podocytes are among the most complex differentiated cells and possess a highly branched array of foot processes that are essential to glomerular filtration in the kidney. Such differentiated podocytes are unable to replicate and culturing of primary podocytes results in rapid growth arrest. Therefore, conditionally immortalized mouse podocyte clones (MPC) were established, which are highly proliferative when cultured under permissive conditions. Nonpermissive conditions render the majority of MPC cells growth arrested within 6 days and induce many characteristics of differentiated podocytes. Both proliferating and differentiating MPC cells express the WT-1 protein and an ordered array of actin fibers and microtubules extends into the forming cellular processes during differentiation, reminiscent of podocyte processes in vivo. These cytoskeletal rearrangements and process formation are accompanied by the onset of synaptopodin synthesis, an actin-associated protein marking specifically differentiated podocytes. In addition, focal contacts are rearranged into an ordered pattern in differentiating MPC cells. Most importantly, electrophysiological studies demonstrate that differentiated MPC cells respond to the vasoactive peptide bradykinin by changes in intracellular calcium concentration. These results suggest a regulatory role of podocytes in glomerular filtration. Taken together, these studies establish that conditionally immortalized MPC cells retain a differentiation potential similar to podocytes in vivo. Therefore, the determinative steps of podocyte differentiation and process formation are studied for the first time using an inducible in vitro model.
Animals↗
Synaptopodin: an actin-associated protein in telencephalic dendrites and renal podocytes.
Synaptopodin is an actin-associated protein of differentiated podocytes that also occurs as part of the actin cytoskeleton of postsynaptic densities (PSD) and associated dendritic spines in a subpopulation of exclusively telencephalic synapses. Amino acid sequences determined in purified rat kidney and forebrain synaptopodin and derived from human and mouse brain cDNA clones show no significant homology to any known protein. In particular, synaptopodin does not contain functional domains found in receptor-clustering PSD proteins. The open reading frame of synaptopodin encodes a polypeptide with a calculated Mr of 73.7 kD (human)/74.0 kD (mouse) and an isoelectric point of 9.38 (human)/9. 27 (mouse). Synaptopodin contains a high amount of proline ( approximately 20%) equally distributed along the protein, thus virtually excluding the formation of any globular domain. Sequence comparison between human and mouse synaptopodin revealed 84% identity at the protein level. In both brain and kidney, in vivo and in vitro, synaptopodin gene expression is differentiation dependent. During postnatal maturation of rat brain, synaptopodin is first detected by Western blot analysis at day 15 and reaches maximum expression in the adult animal. The exclusive synaptopodin synthesis in the telencephalon has been confirmed by in situ hybridization, where synaptopodin mRNA is only found in perikarya of the olfactory bulb, cerebral cortex, striatum, and hippocampus, i.e., the expression is restricted to areas of high synaptic plasticity. From these results and experiments with cultured cells we conclude that synaptopodin represents a novel kind of proline-rich, actin-associated protein that may play a role in modulating actin-based shape and motility of dendritic spines and podocyte foot processes.
Actins↗