Cloning of CDP-Diacylglycerol Synthase from a Human Neuronal Cell Line

A critical step in the supply of substrate for the phosphoinositide signal transduction pathway is the formation of the liponucleotide intermediate, CDP-diacylglycerol, catalyzed by CDP-diacylglycerol synthase. Further insight into the regulation of phosphoinositide biosynthesis was sought by cloning of the gene for the vertebrate enzyme. Sequence of the corresponding gene from Drosophila was used to prepare a probe for screening of a human neuronal cell cDNA library. A cDNA was isolated with a predicted open reading frame of 1,332 bases, encoding a protein of 51 kDa. The amino acid sequence showed 50% identity (75% similarity) to that of Drosophila eye CDP-diacylglycerol synthase and substantial similarity to the Saccharomyces cerevisiae and Escherichia coli homologues. Northern blot analysis, with human cDNA riboprobes, suggested that the corresponding mRNA was expressed in all human tissues examined. Expression of the human cDNA in COS cells resulted in a more than fourfold increase in CDP-diacylglycerol synthase activity. Knowledge of the sequence of vertebrate CDP-diacylglycerol synthase should facilitate further investigations into its regulation and the possible existence of distinct isoforms.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65959/1/j.1471-4159.1996.67052200.x.pd

Biomarker Discovery by Sparse Canonical Correlation Analysis of Complex Clinical Phenotypes of Tuberculosis and Malaria

Biomarker discovery aims to find small subsets of relevant variables in ‘omics data that correlate with the clinical syndromes of interest. Despite the fact that clinical phenotypes are usually characterized by a complex set of clinical parameters, current computational approaches assume univariate targets, e.g. diagnostic classes, against which associations are sought for. We propose an approach based on asymmetrical sparse canonical correlation analysis (SCCA) that finds multivariate correlations between the ‘omics measurements and the complex clinical phenotypes. We correlated plasma proteomics data to multivariate overlapping complex clinical phenotypes from tuberculosis and malaria datasets. We discovered relevant ‘omic biomarkers that have a high correlation to profiles of clinical measurements and are remarkably sparse, containing 1.5–3% of all ‘omic variables. We show that using clinical view projections we obtain remarkable improvements in diagnostic class prediction, up to 11% in tuberculosis and up to 5% in malaria. Our approach finds proteomic-biomarkers that correlate with complex combinations of clinical-biomarkers. Using the clinical-biomarkers improves the accuracy of diagnostic class prediction while not requiring the measurement plasma proteomic profiles of each subject. Our approach makes it feasible to use omics' data to build accurate diagnostic algorithms that can be deployed to community health centres lacking the expensive ‘omics measurement capabilities

Cholesterol Synthesis and Nerve Regeneration

In this report, we examine the requirement of cholesterol biosynthesis and its axonal transport for goldfish optic nerve regeneration. Cholesterol, labeled by intraocular injection of [ 3 H]mevalonolactone. exhibited a delayed appearance in the optic tectum. Squalene and other minor components were labeled but not transported. Following optic nerve crush, the amount of labeled cholesterol transport was elevated, while retinal labeling was not altered relative to control fish. A requirement for cholesterol biosynthesis is inferred from the inhibition of neurite outgrowth in retinal explants caused by the cholesterol synthesis inhibitor, 20, 25-diazacholes-terol. The inhibition of growth could be overcome by addition of mevalonolactone, but not cholesterol, to the medium. Intraperitoneal administration of 200 nmol of dia-zacholesterol resulted in 92-98% inhibition of retinal cholesterol synthesis and accumulation of labeled des-mosterol and other lipids in fish retina and brain which persisted for 2 weeks. Diazacholesterol-treated fish showed no reduction in the amount of lipid-soluble radioactivity transported following intraocular injection of [ 3 H]mevalonolactone, but there were alterations in the chromatographic pattern of the transported labeled lipids. In contrast to its effects on neurite outgrowth in vitro , diazacholesterol did not inhibit optic nerve regeneration in vivo , as measured both by arrival of labeled rapidly transported protein at the tectum and by time required for the return of visual function.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65710/1/j.1471-4159.1984.tb12701.x.pd

Mycobacterium tuberculosis Expresses a Novel Ph-Dependent Divalent Cation Transporter Belonging to the Nramp Family

Mammalian natural resistance–associated macrophage protein (Nramp) homologues are important determinants of susceptibility to infection by diverse intracellular pathogens including mycobacteria. Eukaryotic Nramp homologues transport divalent cations such as Fe2+, Mn2+, Zn2+, and Cu2+. Mycobacterium tuberculosis and Mycobacterium bovis (bacillus Calmette-Guérin [BCG]) also encode an Nramp homologue (Mramp)

Puromycin-induced retention deficit in goldfish as a function of attained training performance level

Goldfish received either 20 or 50 active avoidance training trials followed by puromycin (or no-treatment) and 10 retraining trials either 1 or 7 days following training. While 50 trials resulted in significantly more training avoidances than 20 trials, the groups which received puromycin showed equivalent retention deficits on Day 7. A within-group comparison of fish whose training performance was high or low further revealed that the degree of the retention deficit was independent of achieved training performance level. These data support the hypothesis that puromycin interferes with a memory fixation process that is initiated only upon completion of the training session.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21711/1/0000103.pd

Electroconvulsive shock- or puromycin-induced retention deficits in goldfish given two active-avoidance sessions

In a factorial design, goldfish received 15 active-avoidance training trials on Days 1, 7, and 13 followed by electroconvulsive shock (ECS) or no treatment after the training session on Days 1 and 7. The final retention deficits observed when ECS was given after the first session only or after the second session only were similar. Fish given ECS after both sessions showed a cumulative deficit that approximated the sum of the two effects. Similar results were obtained with puromycin. These data support the hypothesis that ECS and puromycin impair only recent learning. Retrograde amnesia gradients confirmed that the efficacy of these amnestic agents was not altered by a second administration.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21642/1/0000026.pd

A P-type ATPase importer that discriminates between essential and toxic transition metals

Transition metals, although being essential cofactors in many physiological processes, are toxic at elevated concentrations. Among the membrane-embedded transport proteins that maintain appropriate intracellular levels of transition metals are ATP-driven pumps belonging to the P-type ATPase superfamily. These metal transporters may be differentiated according to their substrate specificities, where the majority of pumps can extrude either silver and copper or zinc, cadmium, and lead. In the present report, we have established the substrate specificities of nine previously uncharacterized prokaryotic transition-metal P-type ATPases. We find that all of the newly identified exporters indeed fall into one of the two above-mentioned categories. In addition to these exporters, one importer, Pseudomonas aeruginosa Q9I147, was also identified. This protein, designated HmtA (heavy metal transporter A), exhibited a different substrate recognition profile from the exporters. In vivo metal susceptibility assays, intracellular metal measurements, and transport experiments all suggest that HmtA mediates the uptake of copper and zinc but not of silver, mercury, or cadmium. The substrate selectivity of this importer ensures the high-affinity uptake of essential metals, while avoiding intracellular contamination by their toxic counterparts

The effect of memory blocking antibiotics and their analogs on acetylcholinesterase

The ability of antibiotics to inhibit acetylcholinesterase was measured in homogenates of goldfish brain. Puromycin aminonucleoside was the most potent inhibitor followed by puromycin, cycloheximide and acetoxycycloheximide. Puromycin effectively impaired retention of active-avoidance learning in goldfish when injected either immediately before or after training, while puromycin aminonucleoside did not regardless of injection time. These results suggest that the known amnestic effects of puromycin, cycloheximide and acetoxycycloheximide are not a consequence of interference with acetylcholinesterase.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21729/1/0000121.pd

Magnetic Resonance Imaging with laser polarized 129Xe129Xe

Magnetic Resonance Imaging with laser-polarized 129Xe129Xe can be utilized to trace blood flow and perfusion in tissue for a variety of biomedical applications. Polarized xenon gas introduced in to the lungs dissolves in the blood and is transported to organs such as the brain where it accumulates in the tissue. Spectroscopic studies combined with imaging have been used to produce brain images of 129Xe129Xe in the rat head. This work establishes that nuclear polarization produced in the gas phases survives transport to the brain where it may be imaged. Increases in polarization and delivered volume of 129Xe129Xe will allow clinical measurements of regional blood flow. © 1998 American Institute of Physics.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87439/2/200_1.pd