Biomedical subjects

Warren Zipfel

Publications and source records attributed to Warren Zipfel.

6 recordsLinked to original sources

PubMed2006

BAC transgenic mice express enhanced green fluorescent protein in central and peripheral cholinergic neurons.

The peripheral nervous system has complex and intricate ramifications throughout many target organ systems. To date this system has not been effectively labeled by genetic markers, due largely to inadequate transcriptional specification by minimum promoter constructs. Here we describe transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed under the control of endogenous choline acetyltransferase (ChAT) transcriptional regulatory elements, by knock-in of eGFP within a bacterial artificial chromosome (BAC) spanning the ChAT locus and expression of this construct as a transgene. eGFP is expressed in ChAT(BAC)-eGFP mice in central and peripheral cholinergic neurons, including cell bodies and processes of the somatic motor, somatic sensory, and parasympathetic nervous system in gastrointestinal, respiratory, urogenital, cardiovascular, and other peripheral organ systems. Individual epithelial cells and a subset of lymphocytes within the gastrointestinal and airway mucosa are also labeled, indicating genetic evidence of acetylcholine biosynthesis. Central and peripheral neurons were observed as early as 10.5 days postcoitus in the developing mouse embryo. ChAT(BAC)-eGFP mice allow excellent visualization of all cholinergic elements of the peripheral nervous system, including the submucosal enteric plexus, preganglionic autonomic nerves, and skeletal, cardiac, and smooth muscle neuromuscular junctions. These mice should be useful for in vivo studies of cholinergic neurotransmission and neuromuscular coupling. Moreover, this genetic strategy allows the selective expression and conditional inactivation of genes of interest in cholinergic nerves of the central nervous system and peripheral nervous system.

Animals↗

PubMed2006

In vivo delivery of fluoresceinated dextrans to the murine growth plate: imaging of three vascular routes by multiphoton microscopy.

Bone elongation by endochondral ossification occurs through the differentiation cascade of chondrocytes of cartilaginous growth plates. Molecules from the systemic vasculature reach the growth plate from three different directions: epiphyseal, metaphyseal, and a ring vessel and plexus associated with the perichondrium. This study is an analysis of the real-time dynamics of entrance of fluoresceinated tracers of different molecular weights into the growth plate from the systemic vasculature and tests the hypothesis that molecular weight is a key variable in the determination of both the directionality and the extent of tracer movement into the growth plate. Multiphoton microscopy was used for direct in vivo imaging of the murine proximal tibial growth plate in anesthetized 4- to 5-week-old transgenic mice with green fluorescent protein linked to the collagen II promoter. Mice were given an intracardiac injection of either fluorescein (332.3 Da) or fluoresceinated dextrans of 3, 10, 40, 70 kDa, singly or sequentially. For each tracer, directionality and rate of arrival, together with extent of movement within the growth plate, were imaged in real time. For small molecules (up to 10 kDa), vascular access from all three directions was observed and entrance was equally permissive from the metaphyseal and the epiphyseal sides. Within our detection limit (a few percent of vascular concentration), 40 kDa and larger dextrans did not enter. These results have implications both for understanding systemic and paracrine regulation of growth plate chondrocytic differentiation, as well as variables associated with effective drug delivery to growth plate chondrocytes.

Animals↗

PubMed2006

Ca2+ -induced Ca2+ release through localized Ca2+ uncaging in smooth muscle.

Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) occurs in smooth muscle as spontaneous SR Ca(2+) release or Ca(2+) sparks and, in some spiking tissues, as Ca(2+) release that is triggered by the activation of sarcolemmal Ca(2+) channels. Both processes display spatial localization in that release occurs at a higher frequency at specific subcellular regions. We have used two-photon flash photolysis (TPFP) of caged Ca(2+) (DMNP-EDTA) in Fluo-4-loaded urinary bladder smooth muscle cells to determine the extent to which spatially localized increases in Ca(2+) activate SR release and to further understand the molecular and biophysical processes underlying CICR. TPFP resulted in localized Ca(2+) release in the form of Ca(2+) sparks and Ca(2+) waves that were distinguishable from increases in Ca(2+) associated with Ca(2+) uncaging, unequivocally demonstrating that Ca(2+) release occurs subsequent to a localized rise in [Ca(2+)](i). TPFP-triggered Ca(2+) release was not constrained to a few discharge regions but could be activated at all areas of the cell, with release usually occurring at or within several microns of the site of photolysis. As expected, the process of CICR was dominated by ryanodine receptor (RYR) activity, as ryanodine abolished individual Ca(2+) sparks and evoked release with different threshold and kinetics in FKBP12.6-null cells. However, TPFP CICR was not completely inhibited by ryanodine; Ca(2+) release with distinct kinetic features occurred with a higher TPFP threshold in the presence of ryanodine. This high threshold release was blocked by xestospongin C, and the pharmacological sensitivity and kinetics were consistent with CICR release at high local [Ca(2+)](i) through inositol trisphosphate (InsP(3)) receptors (InsP(3)Rs). We conclude that CICR activated by localized Ca(2+) release bears essential similarities to those observed by the activation of I(Ca) (i.e., major dependence on the type 2 RYR), that the release is not spatially constrained to a few specific subcellular regions, and that Ca(2+) release through InsP(3)R can occur at high local [Ca(2+)](i).

Animals↗

PubMed2004

Liver fatty acid-binding protein gene ablation inhibits branched-chain fatty acid metabolism in cultured primary hepatocytes.

Whereas the role of liver fatty acid-binding protein (L-FABP) in the uptake, transport, mitochondrial oxidation, and esterification of normal straight-chain fatty acids has been studied extensively, almost nothing is known regarding the function of L-FABP in peroxisomal oxidation and metabolism of branched-chain fatty acids. Therefore, phytanic acid (most common dietary branched-chain fatty acid) was chosen to address these issues in cultured primary hepatocytes isolated from livers of L-FABP gene-ablated (-/-) and wild type (+/+) mice. These studies provided three new insights: First, L-FABP gene ablation reduced maximal, but not initial, uptake of phytanic acid 3.2-fold. Initial uptake of phytanic acid uptake was unaltered apparently due to concomitant 5.3-, 1.6-, and 1.4-fold up-regulation of plasma membrane fatty acid transporter/translocase proteins (glutamic-oxaloacetic transaminase, fatty acid transport protein, and fatty acid translocase, respectively). Second, L-FABP gene ablation inhibited phytanic acid peroxisomal oxidation and microsomal esterification. These effects were consistent with reduced cytoplasmic fatty acid transport as evidenced by multiphoton fluorescence photobleaching recovery, where L-FABP gene ablation reduced the cytoplasmic, but not membrane, diffusional component of NBD-stearic acid movement 2-fold. Third, lipid analysis of the L-FABP gene-ablated hepatocytes revealed an altered fatty acid phenotype. Free fatty acid and triglyceride levels were decreased 1.9- and 1.6-fold, respectively. In summary, results with cultured primary hepatocytes isolated from L-FABP (+/+) and L-FABP (-/-) mice demonstrated for the first time a physiological role of L-FABP in the uptake and metabolism of branched-chain fatty acids.

Animals↗

PubMed2002

DNA fragment sizing by single molecule detection in submicrometer-sized closed fluidic channels.

The fabrication of fluidic channels with dimensions smaller than 1 microm is described and characterized in respect to their use for detection of individual DNA molecules. The sacrificial layer technique is used to fabricate these devices as it provides CMOS-compatible materials exhibiting low fluorescence background. It also allows creating microfluidics circuitry of submicrometer dimensions with great control. The small dimensions facilitate single molecule detection and minimize events of simultaneous passage of more than one molecule through the measurement volume. The behavior of DNA molecules inside these channels under an applied electrical field was first studied by fluorescence correlation spectroscopy using M13 double-stranded DNA. A linear relationship between the flow speed and applied electric field across the channel was observed. Speeds as high as 5 mm/s were reached, corresponding to only a few milliseconds of analysis time per molecule. The channels were then used to characterize a mixture of nine DNA fragments. Both the distribution and relative proportions of the individual fragments, as well as the overall concentration of the DNA sample, can be deduced from a single experiment. The amount of sample required for the analysis was approximately 10,000 molecules, or 76 fg. Other potential applications of these submicrometer structures for DNA analysis are discussed.

DNA↗

PubMed2002

Translocation and utilization of fungal storage lipid in the arbuscular mycorrhizal symbiosis.

The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Carbon is transferred from the plant to the fungus as hexose, but the main form of carbon stored by the mycobiont at all stages of its life cycle is triacylglycerol. Previous isotopic labeling experiments showed that the fungus exports this storage lipid from the intraradical mycelium (IRM) to the extraradical mycelium (ERM). Here, in vivo multiphoton microscopy was used to observe the movement of lipid bodies through the fungal colony and to determine their sizes, distribution, and velocities. The distribution of lipid bodies along fungal hyphae suggests that they are progressively consumed as they move toward growing tips. We report the isolation and measurements of expression of an AM fungal expressed sequence tag that encodes a putative acyl-coenzyme A dehydrogenase; its deduced amino acid sequence suggests that it may function in the anabolic flux of carbon from lipid to carbohydrate. Time-lapse image sequences show lipid bodies moving in both directions along hyphae and nuclear magnetic resonance analysis of labeling patterns after supplying 13C-labeled glycerol to either extraradical hyphae or colonized roots shows that there is indeed significant bidirectional translocation between IRM and ERM. We conclude that large amounts of lipid are translocated within the AM fungal colony and that, whereas net movement is from the IRM to the ERM, there is also substantial recirculation throughout the fungus.

Acyl-CoA Dehydrogenase↗