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D J Fink

Publications and source records attributed to D J Fink.

At least 55 records · Page 3Linked to original sources
PubMed1995

Na,K-ATPase beta subunit isoform expression in the peripheral nervous system of the rat.

Using the RNase protection assay (RPA) to study the distribution of isoforms of the non-catalytic (beta) subunit of Na,K-ATPase in the peripheral nervous system, we found both beta 1 and beta 2 isoform mRNAs in dorsal root ganglion (DRG), but only beta 2 mRNA in sciatic nerve. Using Western blot to measure accumulation of the polypeptides at a ligature on the nerve we found that beta 1 but not beta 2 polypeptide is carried by rapid axonal transport in the sciatic nerve. These results imply that beta 1 is the prominent isoform of Na,K-ATPase in neurons and beta 2 the prominent isoform in Schwann cells.

Animals↗
PubMed1995

HSV as a gene transfer vector for the nervous system.

Gene therapy for diseases of the nervous system requires vectors capable of delivering the therapeutic gene into postmitotic cells in vivo. Herpes simplex virus type 1 is a neurotropic virus that naturally establishes latency in neurons of the peripheral nervous system. Replication defective HSV vectors have been developed; these are deleted for at least one essential immediate early regulatory gene, rendering the virus less cytotoxic, incapable of reactivation, but still capable of establishing latency. Foreign genes can be vigorously expressed from an HSV-based vector in a transient manner in brain and other tissues. Long-term but weak foreign gene expression may be achieved in the nervous system by exploiting the transcriptional control mechanisms of the natural viral latency active promoter. To meet the needs of specific applications, either highly active long-term or regulatable transgene expression will be needed, requiring further studies in order to design the appropriate latency-based promoter systems.

Gene Transfer Techniques↗
PubMed1995

Crystallization studies on avian eggshell membranes: implications for the molecular factors controlling eggshell formation.

The avian eggshell is a natural biopolymer and mineral composite. It is a very useful model for biomimetic mineralization, since it is among the fastest forming hard tissues known. Isolated eggshell membranes, which were demineralized in vitro, were used to investigate the in vitro modulation of CaCO3 crystal deposition by organic matrix materials. Crystallization on the demineralized eggshell membrane occurred almost exclusively at the peripheries of residual calcium reserve assemblies, which contain a high concentration of sulfur. Similar structures are observed for eggshell membranes after natural demineralization. The characteristic rhombohedral crystal morphologies of the calcite crystals grown in this in vitro system are much less regular when grown in the presence of organic matrix or partially purified dermatan sulfate proteoglycans obtained from the eggshell. The effect of these macromolecules on the morphology and size of CaCO3 crystals is concentration-dependent. These studies indicate the complexity of the molecular and ionic interactions involved in the initiation and formation of the eggshell, with the focus on the role of the organic matrix.

Animals↗
PubMed1995

Development and application of herpes simplex virus vectors for human gene therapy.

Advances in understanding the molecular basis of human disease and the development of recombinant DNA methods is rapidly creating new means of disease diagnosis and treatment. Among the most revolutionary developments are technologies for transfer of therapeutic genes to the human body to treat both inherited and acquired disease. Gene therapy offers considerable promise for ameliorating otherwise intractable diseases such as immunopathological conditions, cancer, heart disease, and various metabolic and neurodegenerative syndromes. To fulfill this promise, more efficient and effective methods of gene delivery and appropriate gene expression must be developed. The lack of such techniques is currently the most significant impediment to the use of genetic therapy. Both viral and nonviral delivery systems are under development for specific gene-therapy applications. Herpes simplex virus (HSV) represents a novel vector system for gene delivery to the nervous system and other tissues. HSV is able to establish latency in nondividing neuronal cells in which genomes persist long-term but do not integrate or alter host-cell metabolism and that carry a promoter system uniquely capable of escaping repression that shuts off the expression of HSV-lytic genes during latency. This review examines efforts to create defective HSV vectors that are safe, noncytotoxic, and applicable to the treatment of cancer and diseases affecting peripheral nerves. Perhaps the most important use of HSV vectors will be for the treatment of neurodegenerative diseases of the brain, but additional studies are required to improve the design of promoters to ensure regulatable or effective levels of therapeutic gene expression.

Brain Diseases↗
PubMed1994

Gene transfer to brain using herpes simplex virus vectors.

Herpes simplex virus type 1 represents an ideal candidate for development as a vehicle for gene transfer to postmitotic neurons of the central nervous system. The natural biology of this virus makes it well suited for this purpose as it is capable of infecting a variety of neuronal cell types in the brain where the viral genome can persist indefinitely in a latent state. In latency, the viral lytic genes are transcriptionally silent and a unique set of latency-associated transcripts are expressed. Two impediments to using herpes simplex virus vectors must be overcome: (1) A noncytotoxic mutant virus backbone must be engineered, and (2) a suitable promoter-regulator that stably expresses foreign genes from the vector genome during latency must be constructed. Deletion of specific immediate early genes from the vector can render the virus nontoxic to neurons in culture and in vivo following stereotactic inoculation into specific regions of the brain. Because these viruses cannot replicate, they enter latency on infection of central nervous system neurons. A number of viral and cellular promoters have been tested for their ability to express genes during latency. Strong viral promoters and neurospecific promoters display transient activity. Although the promoter regions for the latency-associated transcripts are highly active in the peripheral nervous system, they show low-level but persistent activity in the brain. Experiments are in progress to exploit RNA polymerase III gene promoters or novel recombinant promoters capable of auto-inducing their own expression in order to increase gene expression during latency in brain neurons.

Animals↗
PubMed1994

Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy.

Bone formation in the embryo, and during adult fracture repair and remodeling, involves the progeny of a small number of cells called mesenchymal stem cells (MSCs). These cells continuously replicate themselves, while a portion become committed to mesenchymal cell lineages such as bone, cartilage, tendon, ligament, and muscle. The differentiation of these cells, within each lineage, is a complex multistep pathway involving discrete cellular transitions much like that which occurs during hematopoiesis. Progression from one stage to the next depends on the presence of specific bioactive factors, nutrients, and other environmental cues whose exquisitely controlled contributions orchestrate the entire differentiation phenomenon. An understanding of the cellular and molecular events of osteogenic differentiation of MSCs provides the foundation for the emergence of a new therapeutic technology for cell therapy. The isolation and in vitro mitotic expansion of autologous human MSCs will support the development of novel protocols for the treatment of many clinically challenging conditions. For example, local bone defects can be repaired through site-directed delivery of MSCs in an appropriate carrier vehicle. Generalized conditions, such as osteoporosis, may be treatable by systemic administration of culture-expanded autologous MSCs or through biopharmaceutical regimens based on the discovery of critical regulatory molecules in the differentiation process. With this in mind, we can begin to explore therapeutic options that have never before been available.

Animals↗
PubMed1994

Isoform specific reductions in Na+,K(+)-ATPase catalytic (alpha) subunits in the nerve of rats with streptozotocin-induced diabetes.

Na+,K(+)-ATPase activity in nerve is reduced in rats with streptozotocin-induced diabetes; three different isoforms of the alpha (catalytic) subunit of the enzyme are present in nerve. Using western blot to determine subunit isoform polypeptide levels in sciatic nerve, we found a substantial reduction in alpha 1-isoform polypeptide (88% at 3 weeks, 94% at 8 weeks) after induction of diabetes by streptozotocin. Reductions in alpha 2 and alpha 3 polypeptide were smaller and not statistically significant. The reduction in amount of all three isoform polypeptides in the nerve of 3-week diabetic animals was corrected by administration of insulin. Accumulation of alpha 1 polypeptide at a nerve ligature indicated that rapid transport of that polypeptide in nerve occurs with normal kinetics. The results implicate a specific marked deficit in alpha 1, much more than alpha 2 or alpha 3, catalytic subunit isoform of Na+,K(+)-ATPase in the pathogenesis of diabetic neuropathy.

Animals↗
PubMed1994

PCR-based analysis of herpes simplex virus type 1 latency in the rat trigeminal ganglion established with a ribonucleotide reductase-deficient mutant.

Competitive quantitative PCR and reverse transcriptase-PCR were used to quantitate DNA and RNA from an attenuated ribonucleotide reductase-deleted herpes simplex virus type 1 (HSV-1) mutant in the rat trigeminal ganglion after peripheral inoculation following corneal scarification. Amplification of ganglionic DNA with oligonucleotide primers specific for the HSV-1 glycoprotein B (gB) gene and for the latency-associated transcript (LAT) gene indicated that there were approximately 2 x 10(5) genome equivalents per ganglion at 2 days, 7 days, and 8 weeks after inoculation. Amplification of ganglionic RNA with primers specific for HSV-1 LAT indicated that the amount of LAT RNA was also stable over 8 weeks, with 10(7) LAT molecules per ganglion at 2 days and at 7 days postinoculation and 1.4 x 10(7) LAT molecules per ganglion at 8 weeks. In situ hybridization with a digoxigenin-labeled riboprobe specific for LAT detected an average of one to two LAT-positive cells in each positive 6-microns section of trigeminal ganglion. In situ PCR detection of HSV-1 genomes in similar sections, using digoxigenin-labeled nucleotides with primers specific for HSV-1 gB, identified as many as 120 genome-positive cells per section. These results indicate that there are approximately 50 LAT molecules per latent HSV-1 genome in the trigeminal ganglion, compared with 15 LAT molecules per latent HSV-1 genome in the central nervous system (R. Ramakrishnan, D. J. Fink, G. Jiang, P. Desai, J. C. Glorioso, and M. Levine, J. Virol. 68:1864-1873, 1994), but that cells with detectable LATs by in situ hybridization represent only a small proportion of those ganglionic neurons containing HSV-1 genomes. The presence of latent HSV-1 genomes in a large number of neurons suggests that HSV-1 may be more efficient in establishing the latent state than would be anticipated from previous reports.

Animals↗
PubMed1994

Competitive quantitative PCR analysis of herpes simplex virus type 1 DNA and latency-associated transcript RNA in latently infected cells of the rat brain.

Competitive quantitative PCRs were used to examine the consequences of stereotactically injecting a highly attenuated herpes simplex virus type 1 mutant into rat brains. This mutant virus, designated RR1CAT/RR2lacZ, was engineered so that coding sequences of the genes UL39 and UL40 specifying the subunits of the viral ribonucleotide reductase were replaced by the chloramphenicol acetyltransferase (CAT) and the lacZ gene coding sequences, respectively. Stereotactic injection of this virus into the hippocampal region of the rat brain resulted in a localized infection. Viral gene products were visualized by immunochemical, cytochemical, or in situ hybridization techniques in the injected hippocampal region at 2 days postinjection. Viral genomes, represented by glycoprotein B (gB), latency-associated transcript (LAT), and lacZ sequences could be amplified by PCR from templates obtained by scraping hippocampal tissue off single 10-microns frozen sections. Both gB message and LAT could be detected by reverse transcriptase (RT)-PCR. At day 7 postinjection, neither CAT message, gB message, nor beta-galactosidase activity could be visualized by the same techniques, although viral DNA was detected by PCR and LAT could be detected by RT-PCR. A similar pattern was seen at 8 weeks, suggesting that latency was established by the mutant virus in cells of the injected hippocampus. By competitive quantitative PCR, hippocampal sections were determined to contain 2.6 x 10(5) genome equivalents (represented by the gB gene) on day 2, 6.2 x 10(4) on day 7, and 8.3 x 10(4) at 8 weeks. By competitive quantitative RT-PCR, the numbers of LAT molecules at the same time points were 3.2 x 10(6), 1.3 x 10(6), and 1.2 x 10(6), respectively. The numbers of LAT molecules per genome equivalent were 12.5, 20.3, and 14.5, respectively, being approximately the same for each of the three time points. The data permit the conclusion that the RR mutant virus establishes latency in the rat brain with the persistence of the viral genome and the production of LAT molecules. Once latency is established, the numbers of viral genomes and LAT RNA molecules remain constant. Thus the competitive quantitative PCR and RT-PCR techniques provide very sensitive and reliable methods to quantitate viral DNA and RNA present in infected tissue.

Animals↗
PubMed1994

A novel latency-active promoter is contained within the herpes simplex virus type 1 UL flanking repeats.

Herpes simplex virus type 1 (HSV-1) expresses a unique series of RNA molecules, the latency-associated transcripts or LATs, during latent infection of neuronal tissues. Previous studies by others have described a TATA box-containing latency-active promoter, referred to here as LAP1, located approximately 700 bp upstream of the 5' end of the major 2.0-kb LAT. In this report, transient gene expression assays were employed to identify a second, novel latency-active promoter (LAP2) present within a region downstream of LAP1 and 5' proximal to the major 2.0-kb LAT. In contrast to LAP1, this promoter lacks a TATA box but possesses cis-acting regulatory elements and other features frequently observed within eukaryotic housekeeping gene promoters. Unlike most other HSV promoters, LAP2 was down-regulated by the viral transcriptional activators ICP4 and ICP0. The majority of LAP2-positive regulatory elements were located within sequences from -257 to -58 relative to the 5' end of the 2.0-kb LAT, and the basal promoter mapped within sequences from -14 to +28. RNase protection experiments demonstrated that chimeric LAT-chloramphenicol acetyltransferase transcripts produced in the transient assays initiated at or near the 5' end of the major 2-kb LAT. Tn5 insertional mutagenesis of the ICP4 regulatory gene determined that down-regulation of LAP2 required the ICP4 transactivating domain and targeted the minimal promoter region as the site of action by ICP4. Replicating recombinant viruses containing a LAP2-lacZ reporter gene cassette in an ectopic site (glycoprotein C locus) were shown to be active in mouse trigeminal ganglia. Taken together, these experiments suggest that the LAT region of the HSV-1 genome contains at least two latency-active promoters which may play different roles in expressing the various LATs. Alternatively, these promoters may comprise a larger promoter-regulatory complex which may influence transcription during latency.

Animals↗
PubMed1994

Herpes simplex virus vectors and gene transfer to brain.

Experiments are in progress to engineer herpes simplex virus type 1 as a gene transfer vector for the nervous system. This virus is well suited for this purpose due to its ability to persist for long periods in a latent state in neurons wherein lytic genes are inactive and a unique viral promoter is capable of inducing transcription from the latent viral genome. The virus can be highly cytotoxic for neurons, however, and thus genes which initiate the lytic cycle must be deleted from the genome in order to force the virus into latency. Candidate genes include the immediately early functions ICP0, ICP27, ICP4 and the virus host shut-off function. The design of a suitable promoter regulator capable of expressing foreign genes during latency must also be developed. Many promoters including strong viral promoters and neuronal-specific cellular promoters have proved to be inadequate due to the transient nature of their activity from the viral vector genome and the viral latency promoter region appears to be weakly active in brain. Current efforts concern the development of auto-regulatable promoters which can remain active even if the viral genome is bound by chromatin.

Base Sequence↗
PubMed1993

Differential axonal transport of individual Na,K-ATPase catalytic (alpha) subunit isoforms in rat sciatic nerve.

Three isoforms of the Na,K-ATPase catalytic (alpha) subunit are present in neurons, demonstrated by in situ hybridization of neurons and Western blot of nerve. We used Western blot with antibodies specific for alpha 1, alpha 2 and alpha 3 peptides to measure the accumulation of individual peptides at a ligature on the sciatic nerve. alpha 1 peptide accumulated with kinetics suggesting rapid axonal transport of that isoform within nerve. alpha 2 and alpha 3 peptides did not accumulate at the ligature. These studies provide insight into the dynamics of axonal Na,K-ATPase isoforms.

Animals↗
PubMed1993

Axotomy increases CNTF receptor mRNA in rat spinal cord.

In order to study the role of ciliary neurotrophic factor (CNTF) and its receptor (CNTF-R) in the response of spinal cord neurons to axotomy, we measured the levels of CNTF mRNA in nerve and CNTF-R mRNA in spinal cord following transection of sciatic nerve, using reverse transcriptase PCR. We found CNTF mRNA levels in the nerve fell and that CNTF-R mRNA levels in spinal cord increased at both 1 and 7 days following transection.

Animals↗
PubMed1993

An X Window system for statlab results reporting.

We have developed a system that receives "stat" results encoded in Health Level Seven from the Laboratory Information System, prints a report in destination Intensive Care Units (ICUs), and captures the data for review in a custom spreadsheet format at color X-terminals located in ICUs. Available services include a reference nomogram plot of arterial blood gas data, printed summaries, automated access to the Clinical Information System and a Medline database, electronic mail, a simulated electronic calculator, and general news and information. Security mechanisms include an audit trail of user activities on the system. Noteworthy technical aspects and non-technical factors impacting success are discussed.

Clinical Laboratory Information Systems↗