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Y Chen

Publications and source records attributed to Y Chen.

At least 1,171 records · Page 65Linked to original sources
PubMed1999

In vitro recoating of reovirus cores with baculovirus-expressed outer-capsid proteins mu1 and sigma3.

Reovirus outer-capsid proteins mu1, sigma3, and sigma1 are thought to be assembled onto nascent core-like particles within infected cells, leading to the production of progeny virions. Consistent with this model, we report the in vitro assembly of baculovirus-expressed mu1 and sigma3 onto purified cores that lack mu1, sigma3, and sigma1. The resulting particles (recoated cores, or r-cores) closely resembled native virions in protein composition (except for lacking cell attachment protein sigma1), buoyant density, and particle morphology by scanning cryoelectron microscopy. Transmission cryoelectron microscopy and image reconstruction of r-cores confirmed that they closely resembled virions in the structure of the outer capsid and revealed that assembly of mu1 and sigma3 onto cores had induced rearrangement of the pentameric lambda2 turrets into a conformation approximating that in virions. r-cores, like virions, underwent proteolytic conversion to particles resembling native ISVPs (infectious subvirion particles) in protein composition, particle morphology, and capacity to permeabilize membranes in vitro. r-cores were 250- to 500-fold more infectious than cores in murine L cells and, like virions but not ISVPs or cores, were inhibited from productively infecting these cells by the presence of either NH4Cl or E-64. The latter results suggest that r-cores and virions used similar routes of entry into L cells, including processing by lysosomal cysteine proteinases, even though the former particles lacked the sigma1 protein. To examine the utility of r-cores for genetic dissections of mu1 functions in reovirus entry, we generated r-cores containing a mutant form of mu1 that had been engineered to resist cleavage at the delta:phi junction during conversion to ISVP-like particles by chymotrypsin in vitro. Despite their deficit in delta:phi cleavage, these ISVP-like particles were fully competent to permeabilize membranes in vitro and to infect L cells in the presence of NH4Cl, providing new evidence that this cleavage is dispensable for productive infection.

Animals↗
PubMed1999

Hec1p, an evolutionarily conserved coiled-coil protein, modulates chromosome segregation through interaction with SMC proteins.

hsHec1p, a Homo sapiens coiled-coil-enriched protein, plays an important role in M-phase progression in mammalian cells. A Saccharomyces cerevisiae protein, identical to Tid3p/Ndc80p and here designated scHec1p, has similarities in structure and biological function to hsHec1p. Budding yeast cells deleted in the scHEC1/NDC80 allele are not viable, but this lethal phenotype can be rescued by hsHEC1 under control of the endogenous scHEC1 promoter. At the nonpermissive temperature, significant mitotic delay, chromosomal missegregation, and decreased viability were observed in yeast cells with temperature-sensitive (ts) alleles of hsHEC1. In the hshec1-113 ts mutant, we found a single-point mutation changing Trp395 to a stop codon, which resulted in the expression of a C-terminally truncated 45-kDa protein. The binding of this mutated protein, hshec1-113p, to five identified hsHec1p-associated proteins was unchanged, while its binding to human SMC1 protein and yeast Smc1p was ts. Hec1p also interacts with Smc2p, and the binding of the mutated hshec1-113p to Smc2p was not ts. Overexpression of either hsHEC1 or scHEC1 suppressed the lethal phenotype of smc1-2 and smc2-6 at nonpermissive temperatures, suggesting that the interactions between Hec1p and Smc1p and -2p are biologically significant. These results suggest that Hec1 proteins play a critical role in modulating chromosomal segregation, in part, through their interactions with SMC proteins.

Alleles↗
PubMed1999

Analysis of primary structural determinants that distinguish the centromere-specific function of histone variant Cse4p from histone H3.

Cse4p is a variant of histone H3 that has an essential role in chromosome segregation and centromere chromatin structure in budding yeast. Cse4p has a unique 135-amino-acid N terminus and a C-terminal histone-fold domain that is more than 60% identical to histone H3 and the mammalian centromere protein CENP-A. Cse4p and CENP-A have biochemical properties similar to H3 and probably replace H3 in centromere-specific nucleosomes in yeasts and mammals, respectively. In order to identify regions of Cse4p that distinguish it from H3 and confer centromere function, a systematic site-directed mutational analysis was performed. Nested deletions of the Cse4p N terminus showed that this region of the protein contains at least one essential domain. The C-terminal histone-fold domain of Cse4p was analyzed by changing Cse4p amino acids that differ between Cse4p and H3 to the analogous H3 residues. Extensive substitution of contiguous Cse4p residues with H3 counterparts resulted in cell lethality. However, all large lethal substitution alleles could be subdivided into smaller viable alleles, many of which caused elevated rates of mitotic chromosome loss. The results indicate that residues critical for wild-type Cse4p function and high-fidelity chromosome transmission are distributed across the entire histone-fold domain. Our findings are discussed in the context of the known structure of H3 within the nucleosome and compared with previous results reported for CENP-A.

Amino Acid Sequence↗
PubMed1999

Interaction of angiotensin II and the insulin-like growth factor system in vascular smooth muscle cells.

We studied the effects and interactions of ANG II and the insulin-like growth factor (IGF) system in cultured rat aortic smooth muscle cells. ANG II (1 microM) and IGF-I (10 nM) stimulated both DNA and protein synthesis. The effects of the two peptides in combination were additive or more than additive. The AT(1) receptor antagonist losartan (10 and 100 microM) blocked their synergistic effect on DNA synthesis. IGF binding protein (IGFBP)-1 inhibited the effect of IGF-I but not that of ANG II on DNA synthesis. IGF-I stimulated gene expression of IGFBP-2 and IGFBP-4. ANG II decreased IGF-I, IGFBP-2, and IGFBP-4 transcripts but increased the IGF-I receptor transcript. IGF-I and ANG II in combination had similar effects on gene expression as ANG II alone. The IGFBP-2 and IGFBP-4 peptides could be detected in the conditioned medium. Our results show that ANG II and IGF-I have synergistic effects on vascular smooth muscle cells and that they interact in several ways.

Angiotensin II↗
PubMed1999

A quantitative analysis of cells in the ganglion cell layer of the chick retina.

This study investigated the organization of cells in the ganglion cell layer (GCL) using Nissl staining, retrograde cell degeneration with axotomy of the optic nerve, and retrograde cell labeling by injections of horseradish peroxidase (HRP) into the optic nerve of chicks (posthatching day 1 and 8, P-1 and P-8). The total number of cells in the GCL was 6.1 x 10(6) (P-1) and 4.9 x 10(6) (P-8), and the cell density was 14,300 cells/mm2 (P-1) and 10,400 cells/ mm2 (P-8) on average. Two high-density areas, the central area (CA) and the dorsal area (DA), were observed in the central and dorsal retinas in both P-1 (22,000 cells/mm2 in CA, 19,000 cells/mm2 in DA) and P-8 chicks (19,000 cells/mm2 in CA, 12,800 cells/mm2 in DA). The cell densities in the temporal periphery (TP) and the nasal (NP) peripheral retinas were 7,800 cells/mm2 and 12,500 cells/mm2, respectively, in P-1 and 5,000 cells/ mm2 and 8,000 cells/mm2, respectively, in P-8 chicks. The cell density in the temporal periphery was 35% (P-8) lower than in the nasal periphery in both P-1 and P-8 chicks. Thirty percent (1.9 x 10(6) cells in P-1) of the total cells in the GCL were resistant to axotomy of the optic nerve. The distribution of the axotomy-resistant cells showed two high-density areas in the central and dorsal retinas, corresponding to the CA (5,800 cells/mm2) and the DA (3,200 cells/mm2). These cells also exhibited a center-peripheral increase (2,200 cells/mm2 in the TP) in P-1 chicks, but the high-density area was not found in the dorsal retina of P-8 chicks. From these data and the HRP study, the number of presumptive ganglion cells in P-8 chicks was estimated to be 4 x 10(6) (8,600 cells/mm2 on average), and the density in each area was 13,500 (CA), 10,200 (DA), and 4,300 (TP) cells/mm2. The peripheral/ center ratios of the density of ganglion cells were significantly different along the nasotemporal and dorsoventral axes. The density of ganglion cells decreased more rapidly toward the temporal periphery (TP/CA ratio: 0.47 in P-1 and 0.32 in P-8) than toward the nasal periphery (NP/CA ratio: 0.67 in P-1 and 0.52 in P-8). In contrast, there was no significant difference in the peripheral/center ratios between the dorsal retina (DP/CA ratio: 0.6 in P-1 and 0.56 in P-8) and ventral retina (VP/CA ratio: 0.58 in P-1 and 0.51 in P-8). A small peak in the density of the presumptive ganglion cells was detected in the dorsal retina of both P-1 chicks (10,800 cells/mm2) and P-8 chicks (10,200 cells/mm2). The HRP-labeled cells were small in the CA (M +/- SD: 35.7 +/- 9.1 microm2) and DA (40.0 +/- 11.3 microm2), and their sizes increased toward the periphery (63.4 +/- 29.7 microm2 in the TP) accompanied by a decrease in the cell density. However, the axotomy-resistant cells did not significantly increase in size toward the peripheral retina (12.2 +/- 2.2 microm2 in the CA, 15.2 +/- 3.2 microm2 in the DA, 15.1 +/- 3.8 microm2 in the TP). The characteristic distribution of ganglion cells could be related to visual behavior based upon the specialization of avian visual fields.

Animals↗
PubMed1999

Effect of trilinolein on strophanthidin-induced ventricular tachycardia in guinea pigs.

Trilinolein, a triacylglycerol with linoleic acid, was found to inhibit Ca2+ influx in cardiomyocytes. The purpose of this study was to evaluate the effects of trilinolein on ventricular arrhythmia induced by intravenous strophanthidin in guinea pigs anesthetized with intraperitoneal urethane. After strophanthidin- induced ventricular tachycardia, treatment with trilinolein (0.1, 1, 10 and 100 microg/kg) or control (0.04% propylene glycol) did not terminate ventricular tachycardia. However, 1, 10 and 100 microg/kg trilinolein could narrow the width of the QRS complex during ventricular tachycardia. Pretreatment with trilinolein before strophanthidin administration did not prevent the occurrence of ventricular tachycardia; the doses of strophanthidin required to induce arrhythmias (ventricular extrasystole and ventricular tachycardia) were similar in guinea pigs pretreated with trilinolein or control. However, there were fewer ventricular extrasystoles in guinea pigs pretreated with trilinolein than in the control group (ANOVA, p < 0.01). Moreover, the ventricular extrasystoles were fewer in guinea pigs pretreated with higher doses of trilinolein (100 microg/kg, 103 +/- 60; 10 microg/kg, 188 +/- 86) than lower doses of trilinolein (1 microg/kg, 366 +/- 102; 0.1 microg/kg, 436 +/- 145). This study demonstrated that trilinolein was not effective in terminating or preventing strophanthidin-induced ventricular tachycardia. However, trilinolein could improve ventricular depolarization and suppress ventricular extrasystoles.

Analysis of Variance↗
PubMed1999

Changes of superoxide dismutase in cultured rat aortic smooth muscle cells (A7r5) by an incubation of vitamin E.

Supplementation of antioxidants such as vitamin E and vitamin C as health promotion food is popular recently. Epidemiological studies supported the beneficial effect of these antioxidants because oxygen free radicals have been linked to the process of diseases and aging. The present study evaluated the effect of alpha-tocopherol (vitamin E) on the changes of superoxide dismutase (SOD) in cultured rat aortic smooth muscle cells (A7r5) after a short-term (2 days) or long-term (7 days) incubation. Incubation of A7r5 cells with vitamin E at a concentration of 50 micromol/l for 2 days caused an increase of both the activity and mRNA level of SOD. At higher concentrations, such as 100 or 200 micromol/l, vitamin E failed to enhance SOD more effectively. However, after incubation for 7 days, vitamin E caused a decrease in both the activity and mRNA level of SOD in a concentration-dependent manner. Otherwise, the protein amount of SOD remained the same in these samples regardless of the concentration of vitamin E or the duration of incubation. The obtained results suggest that vitamin E can increase the effect of SOD to result in the beneficial influence of this antioxidant only at low concentration under a short-term supplementation because a down-regulation of SOD was observed in cells receiving a long-term incubation.

Animals↗
PubMed1999

Measurement of sputum Mycobacterium tuberculosis messenger RNA as a surrogate for response to chemotherapy.

Effective treatment regimens for pulmonary tuberculosis are difficult to assess because of the slow growth rate of Mycobacterium tuberculosis in culture and its protracted clearance from sputum. A rapid method that reflects effective antimicrobial activity would markedly advance evaluation of treatment and promote the assessment of new antituberculosis drugs. Conventional methods measure the progressive reduction of numbers of acid-fast bacilli in the sputum smear and the clearance of organisms in sputum culture. In this study, we measured levels of M. tuberculosis 85B (alpha antigen) messenger RNA (mRNA), 16S ribosomal RNA (rRNA), and IS6110 DNA in patients' sputa to ascertain whether they could serve as potential surrogate markers of response to chemotherapy. Sputum specimens were sequentially collected for up to a year from 19 smear-positive pulmonary tuberculosis patients receiving an optimal drug treatment regimen. Nucleic acids were isolated from these specimens, and two M. tuberculosis molecular targets (mRNA, DNA) were quantified, using the ABI Prism 7700 Sequence Detection System. The Mycobacterium genus-specific 16S rRNA was quantified with a limiting dilution RT-PCR assay. Results show that levels of 85B mRNA declined after initiation of therapy, as did viable M. tuberculosis colony counts, with 90% of patients becoming negative for both markers after 2 mo of treatment. The rapid disappearance of M. tuberculosis mRNA from sputum suggests that it is a good indicator of microbial viability and a useful marker for rapid assessment of response to chemotherapy.

Adult↗
PubMed1999

Activation of the insulin-like growth factor-binding protein-5 promoter in osteoblasts by cooperative E box, CCAAT enhancer-binding protein, and nuclear factor-1 deoxyribonucleic acid-binding sequences.

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) has IGF-dependent and -independent actions. PGE2 rapidly increases IGFBP-5 expression by osteoblasts through cAMP-dependent processes. A minimal DNA sequence required for basal and PGE2-stimulated IGFBP-5 promoter activity spans -69 to -35 bp. This region adjoins a functional TATA box and contains E box, CCAAT enhancer-binding protein (C/EBP), nuclear factor-1 (NF-1), and activator protein-2 (AP-2) transcription factor related binding motifs. In this study we compared minimal promoter sequences of -74 to +120 bp, without or with mutations in each potential regulatory element, by reporter gene expression and electrophoretic mobility shift assays. Mutation of the E box-related element reduced basal promoter activity by 50% and eliminated the 2-fold stimulatory effect of PGE2. In contrast, mutations in the C/EBP- or NF-1-related elements also reduced basal promoter activity without fully eliminating the PGE2 effect. Overexpression of C/EBPdelta stimulated basal IGFBP-5 promoter activity, and this effect was eliminated by mutating the C/EBP-binding site. However, mutation of the AP-2-binding site or overexpression of AP-2 did not correlate with basal or PGE2-induced promoter activation. By electrophoretic mobility shift assay, prominent gel shift complexes occurred with osteoblast nuclear extracts and 32P-labeled probes spanning the E box-, C/EBP-, and NF-1-related motifs. These gel shift complexes were depleted by specific binding site mutations and were enhanced by PGE2. Increased binding by extracts from PGE2-treated cultures was blocked by cycloheximide treatment. These results identify several elements as integral binding sequences for both basal and PGE2-stimulated IGFBP-5 promoter activity. They further reveal that multiple sequences within this cluster form a basic transcription unit where nuclear factors can accumulate in a protein synthesis-dependent way and enhance IGFBP-5 expression by osteoblasts in response to PGE2.

Animals↗
PubMed1999

Role of signal transduction in internalization of the G protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein.

For G protein-coupled receptors, limited information is available on the role of agonist binding or of the second-messenger products of receptor signaling on receptor endocytosis. We explored this problem using the opossum PTH/PTH-related protein (PTHrP) receptor, a prototypical Class II G protein-coupled receptor, as a model. In one approach, we evaluated the endocytic properties of mutated forms of the opossum PTH/PTHrP receptor that we had previously shown to be impaired in their ability to initiate agonist-induced signaling when expressed in COS-7 cells. A point mutation in the third cytoplasmic loop (K382A) that severely impairs PTH/PTHrP receptor signaling significantly reduced internalization, whereas two mutant receptors that displayed only partial defects in signaling were internalized normally. To explore more directly the role of second-messenger pathways, we used a cleavable biotinylation method to assess endocytosis of the wild-type receptor stably expressed in human embryonic kidney (HEK) 293 cells. A low rate of constitutive internalization was detected (<5% over a 30-min incubation at 37 C); the rate of receptor internalization was enhanced about 10-fold by the receptor agonists PTH(1-34) or PTHrP(1-34), whereas the receptor antagonist PTH(7-34) had no effect. Forskolin treatment produced a minimal increase in constitutive receptor endocytosis, and the protein kinase (PK)-A inhibitor H-89 failed to block agonist-stimulated endocytosis. Similarly, activation of PK-C, by treatment with phorbol 12-myristate 13-acetate, elicited only a minimal increase in constitutive receptor endocytosis; and blockade of the PK-C pathway, by treatment with a bisindolylmaleimide, failed to inhibit agonist-induced receptor endocytosis. Immunofluorescence confocal microscopic studies of PTH/PTHrP receptor internalization confirmed the results using receptor biotinylation. These findings suggest that: 1) agonist binding is required for the efficient endocytosis of the PTH/PTHrP receptor; 2) receptor activation (agonist-induced receptor conformational change) and/or coupling to G proteins plays a critical role in receptor internalization; and 3) activation of PK-A and PK-C is neither necessary nor sufficient for agonist-stimulated receptor internalization.

Animals↗
PubMed1999

Roles of pathway-specific and inhibitory Smads in activin receptor signaling.

Activins and other members of the transforming growth factor-beta-like superfamily of growth factors transduce their signals by interacting with two types of receptor serine/threonine kinases. The Smad proteins, a new family of intracellular mediators are involved in the signaling pathways of these receptors, but the initial stages of their activation as well as their specific functions remain to be defined. We report here that the pathway-specific Smad2 and 3 can form a complex with the activin receptor in a ligand-dependent manner. This complex formation is rapid but also transient. Indeed, soon after their association with the activin receptor, Smad2 and Smad3 are released into the cytoplasm where they interact with the common partner Smad4. These Smad complexes then mediate activin-induced transcription. Finally, we show that the inhibitory Smad7 can prevent the association of the two pathway-specific Smads with the activin receptor complex, thereby blocking the activin signal.

Activin Receptors, Type I↗
PubMed1999

Mutants of cubitus interruptus that are independent of PKA regulation are independent of hedgehog signaling.

Hedgehog (HH) is an important morphogen involved in pattern formation during Drosophila embryogenesis and disc development. cubitus interruptus (ci) encodes a transcription factor responsible for transducing the hh signal in the nucleus and activating hh target gene expression. Previous studies have shown that CI exists in two forms: a 75 kDa proteolytic repressor form and a 155 kDa activator form. The ratio of these forms, which is regulated positively by hh signaling and negatively by PKA activity, determines the on/off status of hh target gene expression. In this paper, we demonstrate that the exogenous expression of CI that is mutant for four consensus PKA sites [CI(m1-4)], causes ectopic expression of wingless (wg) in vivo and a phenotype consistent with wg overexpression. Expression of CI(m1-4), but not CI(wt), can rescue the hh mutant phenotype and restore wg expression in hh mutant embryos. When PKA activity is suppressed by expressing a dominant negative PKA mutant, the exogenous expression of CI(wt) results in overexpression of wg and lethality in embryogenesis, defects that are similar to those caused by the exogenous expression of CI(m1-4). In addition, we demonstrate that, in cell culture, the mutation of any one of the three serine-containing PKA sites abolishes the proteolytic processing of CI. We also show that PKA directly phosphorylates the four consensus phosphorylation sites in vitro. Taken together, our results suggest that positive hh and negative PKA regulation of wg gene expression converge on the regulation of CI phosphorylation.

Animals↗
PubMed1999

RNA secondary structure and compensatory evolution.

The classic concept of epistatic fitness interactions between genes has been extended to study interactions within gene regions, especially between nucleotides that are important in maintaining pre-mRNA/mRNA secondary structures. It is shown that the majority of linkage disequilibria found within the Drosophila Adh gene are likely to be caused by epistatic selection operating on RNA secondary structures. A recently proposed method of RNA secondary structure prediction based on DNA sequence comparisons is reviewed and applied to several types of RNAs, including tRNA, rRNA, and mRNA. The patterns of covariation in these RNAs are analyzed based on Kimura's compensatory evolution model. The results suggest that this model describes the substitution process in the pairing regions (helices) of RNA secondary structures well when the helices are evolutionarily conserved and thermodynamically stable, but fails in some other cases. Epistatic selection maintaining pre-mRNA/mRNA secondary structures is compared to weak selective forces that determine features such as base composition and synonymous codon usage. The relationships among these forces and their relative strengths are addressed. Finally, our mutagenesis experiments using the Drosophila Adh locus are reviewed. These experiments analyze long-range compensatory interactions between the 5' and 3' ends of Adh mRNA, the different constraints on secondary structures in introns and exons, and the possible role of secondary structures in RNA splicing.

Alcohol Dehydrogenase↗
PubMed1999

Localization of sympathetic, parasympathetic and sensory neurons innervating the heart of the Beijing duck by means of the retrograde transport of horseradish peroxidase.

Sympathetic, parasympathetic and sensory neurons were labeled by injections of horseradish peroxidase into various regions of the heart in 33 Beijing ducks. Sympathetic postganglionic neurons innervating the heart were located in the paravertebral ganglia C15 (C16 is the last cervical segment in the duck) to T3, especially in the ganglion T1. The coronary sulcus and ventricle were more abundantly innervated by sympathetic neurons than the atrium. The left side of the heart was preferentially innervated by sympathetic postganglionic neurons in the left side of paravertebral ganglia but the right side of the heart were equally supplied from the right and left ganglia. Within the medulla oblongata, the number of labeled vagal preganglionic neurons in the nucleus ambiguus was much greater than that in the dorsal motor nucleus of the vagus nerve. Labeled neurons of the nucleus ambiguus were found in many ducks injected into the coronary sulcus. Cardiac sensory neurons were observed in the dorsal root ganglia C15 to T2 (highest in the ganglion T1) and in the nodose and jugular ganglia of the vagus nerve. These labeled neurons probably form the afferent and efferent limbs of cardiac reflexes and control circulation in the Beijing duck.

Animals↗
PubMed1999

Morphological classification of ganglion cells in the central retina of chicks.

Classification of retinal ganglion cells (RGCs) in the chick central retina was studied by retrograde labeling of carbocyanine dye (DiI) and intracellular filling with Lucifer Yellow. Ganglion cells were divided into 4 groups, Group Ic/Is, Group IIc/IIs, Group IIIs, Group IVc, according to sizes of somal area and dendritic field and dendritic branching pattern. Group I cells had small somal area and small dendritic field. They were further divided into 2 subgroups by complexity (subgroup Ic) and simplicity (subgroup Is) of the dendritic arborization. Group II cells had medium-sized soma and dendritic field. They were also divided into subgroup IIc and IIs by the same definitions as those of subgroup Ic and Is. Group IIIs had medium-sized soma, large and simple dendritic arborization. Group IVc in which all cells had large soma, showed large and complex dendritic arborization. Cell populations of each group were 51.8% (subgroup Ic), 21.1% (subgroup Is), 6.2% (subgroup IIc), 14.6% (subgroup IIs), 4.2% (Group IIIs), and 2.1% (Group IVc). Subgroup Ic cells, which were very similar to beta-cells in the mammalian central area, represented about a half of the ganglion cell population. Cells in subgroup Is and IIs, which were not reported in the mammalian retina, were found in the chick central retina in relatively high population (35.7%). Morphological features of chick RGCs in the central retina were considered in comparison with those of other vertebrates.

Amidines↗
PubMed1999

Matrix vesicle plasma cell membrane glycoprotein-1 regulates mineralization by murine osteoblastic MC3T3 cells.

A naturally occurring nonsense truncation mutation of the inorganic pyrophosphate (PPi)-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) PC-1 is associated with spinal and periarticular ligament hyperostosis and cartilage calcification in "tiptoe walking" (ttw) mice. Thus, we tested the hypothesis that PC-1 acts directly in the extracellular matrix to restrain mineralization. Cultured osteoblastic MC3T3 cells expressed PC-1 mRNA and produced hydroxyapatite deposits at 12-14 days. NTPPPH activity increased steadily over 14 days. Transforming growth factor-beta and 1,25-dihydroxyvitamin D3 increased PC-1 and NTPPPH in matrix vesicles (MVs). Because PC-1/NTPPPH was regulated in mineralizing MC3T3 cells, we stably transfected or infected cells with recombinant adenovirus, in order to express 2- to 6-fold more PC-1. PC-1/NTPPPH and PPi content increased severalfold in MVs derived from cells transfected with PC-1. Furthermore, MC3T3 cells transfected with PC-1 deposited approximately 80-90% less hydroxyapatite (by weight) than cells transfected with empty plasmid or enzymatically inactive PC-1. ATP-dependent 45Ca precipitation by MVs from cells overexpressing active PC-1 was comparably diminished. Thus, regulation of PC-1 controls the PPi content and function of osteoblast-derived MVs and matrix hydroxyapatite deposition. PC-1 may provide a novel therapeutic target in certain disorders of bone mineralization.

Adenoviridae↗
PubMed1999

Novel eicosanoids. Isoprostanes and related compounds.

The discovery of IsoPs has been an interesting development for a number of reasons, apart from the fact that it involves novel biochemistry. The simple fact that prostanoids are produced nonenzymatically in prodigous quantities in vivo and in much greater quantities than prostaglandins generated by the cyclooxygenase enzyme was a remarkable finding. The observation that detectable quantities of F2-IsoPs are present in all tissues and human biological fluids carries interesting implications. Previously, there had been little convincing evidence for the occurrence of lipid peroxidation in vivo except under unusual conditions of severe oxidative stress. However, the finding that F2-IsoPs can be easily detected in normal humans suggests a continuous level of ongoing oxidative injury that is not completely suppressed by the elaborate system of antioxidant defenses that have evolved. Another very important aspect of the discovery of IsoPs is that it has brought to the field a long sought after reliable approach to assess oxidative stress status in vivo. The continuing and expanded use of measurements of IsoPs for this purpose will contribute in a very valuable way to advancing our understanding of the role of free radicals in human disease processes. Further, the finding that these compounds are not simply markers of oxidant injury but can also exert potent biological actions both by interaction with specific receptors and, in the case of IsoLGs and cyclopentenenone IsoPs, by virtue of their chemical reactivity, has identified several new classes of molecules that are produced by free radical-induced lipid peroxidation that may mediate some of the adverse sequela of oxidant injury. The elucidation of the variety of compounds that are produced as products of the IsoP pathway and more recently the NP pathway provides vast new areas for scientific inquiry that should yield new and interesting information as this area continues to advance.

Antioxidants↗