Temporal trends in the use of invasive cardiac procedures for non-ST segment elevation acute coronary syndromes according to initial risk stratification.

BACKGROUND: Current guidelines support an early invasive strategy in the management of high-risk non-ST elevation acute coronary syndromes (NSTE-ACS). Although studies in the 1990s suggested that highrisk patients received less aggressive treatment, there are limited data on the contemporary management patterns of NSTE-ACS in Canada. OBJECTIVE: To examine the in-hospital use of coronary angiography and revascularization in relation to risk among less selected patients with NSTE-ACS. METHODS: Data from the prospective, multicentre Global Registry of Acute Coronary Events (main GRACE and expanded GRACE2) were used. Between June 1999 and September 2007, 7131 patients from across Canada with a final diagnosis of NSTE-ACS were included the study. The study population was stratified into low-, intermediate- and high-risk groups, based on their calculated GRACE risk score (a validated predictor of in-hospital mortality) and according to time of enrollment. RESULTS: While rates of in-hospital death and reinfarction were significantly (P<0.001) greater in higher-risk patients, the in-hospital use of cardiac catheterization in low- (64.7%), intermediate- (60.3%) and highrisk (42.3%) patients showed an inverse relationship (P<0.001). This trend persisted despite the increase in the overall rates of cardiac catheterization over time (47.9% in 1999 to 2003 versus 51.6% in 2004 to 2005 versus 63.8% in 2006 to 2007; P<0.001). After adjusting for confounders, intermediate-risk (adjusted OR 0.80 [95% CI 0.70 to 0.92], P=0.002) and high-risk (adjusted OR 0.38 [95% CI 0.29 to 0.48], P<0.001) patients remained less likely to undergo in-hospital cardiac catheterization. CONCLUSION: Despite the temporal increase in the use of invasive cardiac procedures, they remain paradoxically targeted toward low-risk patients with NSTE-ACS in contemporary practice. This treatment-risk paradox needs to be further addressed to maximize the benefits of invasive therapies in Canada.

Atrophy of Müller glia and photoreceptor cells in chick retina misexpressing cNSCL2.

PURPOSE: To investigate whether and how the basic helix-loop-helix (bHLH) gene cNSCL2 is involved in retinal development. METHODS: cNSCL2, the chick homologue of human NSCL2, was isolated and sequenced. In situ hybridization was used to examine its spatial and temporal expression pattern in the retina. Replication-competent retrovirus RCAS was used to drive cNSCL2 misexpression in the developing chick retina, and the effect of the misexpression was analyzed. RESULTS: Expression of cNSCL2 in the retina was restricted. Its mRNA was detected in amacrine and horizontal cells, but not in photoreceptor, bipolar, or ganglion cells. Retroviral-driven misexpression of cNSCL2 in the developing chick retina resulted in missing photoreceptor cells and gross deficits in the outer nuclear layer (ONL). These deficits were probably not because of decreased photoreceptor production, in that the ONL appeared normal in early developmental stages. TUNEL+ cells were detected in the ONL, indicating that photoreceptor cells underwent apoptosis in retinas misexpressing cNSCL2. Müller glial cells were far fewer in the experimental retina than in the control, indicating that cNSCL2 also caused Müller glia atrophy. The onset of Müller glia disappearance preceded that of photoreceptor degeneration. CONCLUSIONS: Expression of cNSCL2 in the chick retina was restricted to amacrine and horizontal cells. Misexpression of cNSCL2 caused severe retinal degeneration, and photoreceptor cells and Müller glia were particularly affected.

neurogenin2 elicits the genesis of retinal neurons from cultures of nonneural cells.

neurogenin2 (ngn2) encodes a basic helix-loop-helix transcription factor and plays an important role in neurogenesis from migratory neural crest cells. Its role in retinal development is poorly understood. We observed that in the developing chick retina, ngn2 was expressed in a subpopulation of proliferating progenitor cells. Ectopic expression of ngn2 in nonneural, retinal pigment epithelial cell culture triggered de novo generation of cells that expressed neural-specific markers and exhibited neuronal morphologies. Further molecular and morphological analyses showed that the main products of the induced neurogenesis were cells resembling young photoreceptor cells and cells resembling retinal ganglion cells. The generation of multiple cell types suggests that ngn2 induces various retinal pathways. Thus, unlike in the peripheral nervous system where ngn2 specifies one type of sensory neuron, ngn2 in the retina is likely involved in a common step leading to different cellular pathways. Our finding that ngn2 can instruct nonneural retinal pigment epithelial cells to differentiate toward retinal neurons demonstrates one possible way to induce de novo retinal neurogenesis.

Embryonic abnormalities from misexpression of cNSCL1.

cNSCL1 encodes a bHLH transcription factor and is specifically expressed in the developing nervous system. We used a replication-competent retrovirus to drive misexpression of cNSCL1 in chick embryos. We found that cNSCL1 misexpression was embryonic lethal and the embryos exhibited gross abnormalities. Many skeletal bones were abnormal and some were completely absent. Expression of BMP4 was reduced. The abnormalities were due to cNSCL1 misexpression in the systemic region, since microinjection of cNSCL1 retrovirus at one hindlimb primordium severely retarded its development, while other limbs on the same animal appeared normal. Similar misexpression of cNSCL2, a closely related bHLH gene, did not produce these phenotypes. Thus, the detrimental effects on embryonic development were specific to cNSCL1. These data indicate that cNSCL1 expression must be tightly regulated during development.

Differential induction of gene expression by basic fibroblast growth factor and neuroD in cultured retinal pigment epithelial cells.

Embryonic chick retinal pigment epithelial (RPE) cells can undergo transdifferentiation upon appropriate stimulation. For example, basic fibroblast growth factor (bFGF) induces intact RPE tissue younger than embryonic day 4.5 (E4.5) to transdifferentiate into a neural retina. NeuroD, a gene encoding a basic helix-loop-helix transcription factor, triggers de novo production of cells that resemble young photoreceptor cells morphologically and express general neuron markers (HNK-1/N-CAM and MAP2) and a photoreceptor-specific marker (visinin) from cell cultures of dissociated E6 RPE (Yan & Wang, 1998). The present study examined whether bFGF will lead to the same transdifferentiation phenomenon as neuroD when applied to dissociated, cultured E6 RPE cells, and whether interplay exists between the two factors under the culture conditions. Dissociated E6 RPE cells were cultured in the presence or absence of bFGF, and with or without the addition of retrovirus expressing neuroD. Gene expression was analyzed with immunocytochemistry and in situ hybridization. Unlike neuroD, bFGF did not induce the expression of visinin, or HNK-1/N-CAM and MAP2. However, bFGF elicited the expression of RA4 immunogenicity; yet, many of these RA4-positive cells lacked a neuronal morphology. Addition of bFGF to neuroD-expressing cultures did not alter the number of visinin-expressing cells; misexpression of neuroD in bFGF-treated cultures did not change the number of RA4-positive cells, suggesting the absence of interference or synergistic interaction between the two factors. Our data indicated that bFGF and neuroD induced the expression of different genes in cultured RPE cells.

Expression of an array of photoreceptor genes in chick embryonic retinal pigment epithelium cell cultures under the induction of neuroD.

Coaxing plastic, non-neuronal cells to transdifferentiate into a particular type of neurons might have clinical applications. Previously we reported that neuroD induces transdifferentiation of retinal pigment epithelium (RPE) cells derived from day-6 chick embryos into cells that resemble young photoreceptor cells. These cells also express visinin, a gene expressed early during cone photoreceptor differentiation. Further characterization showed that the transdifferentiated cells express a number of photoreceptor genes, including interphotoreceptor retinoid binding protein, the alpha-subunit of phosphodiesterase, and opsin genes encoding rhodopsin, the red, the green, and the blue visual pigments. Our data demonstrate that neuroD can reprogram RPE to become photoreceptor cells with substantial differentiation, and suggest the possibility of generating photoreceptor cells from RPE using neuroD as a molecular trigger.

Misexpression of cNSCL1 disrupts retinal development.

cNSCL1 is the chick homologue of mammalian NSCL1, a basic helix-loop-helix gene transiently expressed during neurogenesis. To gain insight into its function, we studied the involvement of cNSCL1 in retinal neurogenesis. In situ hybridization showed dynamic, cell-type-specific expression of cNSCL1, first in developing ganglion cells and later in glial cells. This is drastically different from the expression of neuroD in young photoreceptor cells and their precursors, demonstrating that the proposed neurogenin --> neuroD --> NSCL1 cascade might not apply to retinal neurogenesis in the chick. Small eyes were produced when cNSCL1 was misexpressed in the retinal neuroepithelium through viral transduction. Pulse-labeling with BrdU and [(3)H]thymidine revealed a significant decrease in cell proliferation activity with cNSCL1 misexpression. Massive cell death occurred, but only after cell proliferation activity had subsided, resulting in major distortions of retinal structure. Our data demonstrate the importance of regulated expression of cNSCL1 during retinal development.

Misexpression of a bHLH gene, cNSCL1, results in abnormal brain development.

NSCL1 is a basic helix-loop-helix transcription factor involved in the development of the nervous system. To elucidate its role in neurogenesis, we cloned chick NSCL1 (cNSCL1) and examined its expression pattern and the effect of its misexpression on brain development. cNSCL1 was predominantly expressed during active neurogenesis. Double-labeling experiments showed that proliferating neuroblasts in the ventricular zone lacked cNSCL1 expression and cells expressing cNSCL1 were located just outside the ventricular zone. Retroviral misexpression of cNSCL1 in chick embryos produced a brain with abnormal structure. While the forebrain of the embryonic day-12 (E12) brain appeared normal, the tectum was enlarged. The enlargement was likely due to an increase in cell proliferation, since more radioactivity was detected in this region of the brain after [3H]thymidine labeling at E9. The cerebellum, on the other hand, was reduced in size. Fewer cells were labeled with BrdU in the external granule layer (a secondary germinal layer required for cerebellum development) in experimental embryos than in the controls, suggesting that misexpression of cNSCL1 might interfere with cell proliferation in the external granular layer. Our data indicate that regulated expression of cNSCL1 is required for normal brain development. They also imply that cNSCL1 might be involved in preventing some postmitotic cells from reentering the cell cycle during neurogenesis. Dev Dyn 1999;215:238-247.

neuroD induces photoreceptor cell overproduction in vivo and de novo generation in vitro.

The molecular mechanisms underlying the generation of the various types of cells in the vertebrate retina are largely unknown. We investigated the possibility that genes belonging to the basic helix-loop-helix (bHLH) family of transcriptional factors participate in cell-type specification during retinal neurogenesis. Chick neuroD was isolated from an embryonic cDNA library and its deduced amino acid sequence showed 75% identity with mouse neuroD. In situ hybridization showed that neuroD was expressed in cells located at the outer portion of the developing retinal neuroepithelium, the location where prospective photoreceptors reside. Misexpression of neuroD in retinal neuroepithelium through replication-competent, transformation-deficient retroviruses produced a retina with three, instead of two, layers of photoreceptor cells; the number of cells that express visinin, a marker for cone photoreceptors, increased over 50% compared to control embryos misexpressing the green fluorescent protein. No significant changes were observed in the number of other retinal neurons, including those that express RA4 (ganglion cells), pax6 (ganglion cells and amacrine cells), and chx10 (bipolar cells). Retroviral-driven misexpression of neuroD in monolayer cultures of retinal pigment epithelium yielded de novo production of photoreceptor cells with no other types of retinal neurons detected. We propose that neuroD is important for photoreceptor cell production in the vertebrate retina.

Identification and characterization of tenp, a gene transiently expressed before overt cell differentiation during neurogenesis.

The initiation of cellular differentiation in the vertebrate central nervous system is a cascade of regulated gene expression events involving both intrinsic and extrinsic factors. Currently, the molecular events underlying the developmental transition from the cessation of cell proliferation to the onset of cell differentiation during neurogenesis are still poorly understood. We have identified a gene, tenp, which is likely to play a role during the transition. tenp mRNA was detected in the developing retina and brain, but not in the heart, kidney, or liver. Anatomically, cells expressing tenp formed a narrow strip at the boundary between the ventricular zone (consisting of proliferating cells) and the intermediate zone (occupied by postmitotic, differentiating cells). Further analysis showed that they were bromodeoxyuridine negative and thus postmitotic, yet without apparent differentiation such as the expression of microtubule-associated protein (MAP2) and neurofilament (NF68). When expressed in chicken embryonic fibroblast cells through transfection, Tenp protein was immunodetected in membrane fractions, implying that Tenp might be a membrane protein as predicted by a computer analysis of its primary sequence. Our data suggested that tenp might be involved in an early neurogenic event that existed transiently after terminal mitosis, yet before the overt differentiation of neural precursor cells.

Increased chromokinesin immunoreactivity in retinoblastoma cells.

chromokinesin is a developmentally down-regulated gene with specific expression in proliferating cells during embryonic chick development. It encodes a DNA-binding motor protein localized along the chromosome arm during mitosis, suggesting that the protein may be a component of the long-observed, yet poorly understood 'ejection force' hypothesized to be involved in controlling the direction and speed of chromosome movement. We have isolated human chromokinesin; with affinity-purified antibodies we demonstrated immunocytochemically that Chromokinesin was present at a much higher level in cultured retinoblastoma cells than in primary cultures of human dermal fibroblasts. The increase in immunoreactivity was particularly prominent in interphase cells, whereas in primary cultures of fibroblasts immunopositive cells were predominantly M-phase cells. These observations imply a deregulation of chromokinesin in retinoblastoma cells. Data presented here may be useful in designing strategies to modulate chromosome movement and cell proliferation with either antisense oligonucleotides or specific antibodies, and hence may set the stage for further investigations of the involvement of chromosome motor molecules in mitosis under normal and pathological conditions.

Enzymatic and biochemical probes of residues external to the translocation pathway of UhpT, the sugar phosphate carrier of Escherichia coli.

Part of the substrate translocation pathway through UhpT, the Escherichia coli sugar phosphate carrier, has been assigned to a transmembrane helix extending between residues 260 and 282. To set limits on the external portion of the pathway, we identified nearby residues fully exposed to the periplasm. In one case, we used Western blots to evaluate cleavage by extracellular trypsin. The protease cleaved UhpT variants retaining lysine 294, but not those lacking lysine 294, indicating that trypsin acts at a single extracellular site, lysine 294. In other work we labeled single-cysteine variants with 3-(N-maleimidylpropionyl)biocytin and scored accessibility to extracellular streptavidin by shifts of SDS-polyacrylamide gel electrophoresis mobility. Positions 283 and 284 were fully exposed to the periplasm, since the modified residue was bound by streptavidin in the native protein; by contrast, although the biotin-linked probe modified position 276, streptavidin decoration was not achieved without protein denaturation. We conclude that a 12-residue stretch(283-294) of UhpT is sufficiently exposed to be accessible to large probes (trypsin, streptavidin), while position 276 and more proximal residues are more deeply buried or otherwise shielded from the external phase.

Residues in the pathway through a membrane transporter.

The structure of solute transporters is understood largely from analysis of their amino acid sequences, and more direct information is greatly needed. Here we report work that applies cysteine scanning mutagenesis to describe structure-function relations in UhpT, a bacterial membrane transporter. By using an impermeant SH-reactive agent to probe single-cysteine variants, we show that UhpT transmembrane segment 7 spans the membrane as an alpha-helix and that the central portion of this helix is exposed to both membrane surfaces, forming part of the translocation pathway through this transporter.

Bacterial anion exchange: reductionist and integrative approaches to membrane biology.

Studies of two different bacterial anion exchange proteins (antiporters) led us to conclude that both reductionist and integrative approaches contribute to progress in understanding membrane biology. We have used a reductionist perspective in applying cysteine scanning mutagenesis to probe individual amino acid positions of UhpT (uptake of hexose phosphate transporter), the carrier responsible for transport of glucose 6-phosphate by Escherichia coli. This work has established experimental criteria that should allow one to identify and localize the translocation pathway in such membrane proteins. An integrative view is exemplified by work with OxlT (oxalate transporter), the carrier used by an anaerobe Oxalobacter formigenes to catalyze the antiport of divalent oxalate and monovalent formate. The activity of OxlT is functionally coordinated with that of a cytosolic oxalyl decarboxylase; together, these vectorial and scalar activities constitute a metabolic proton pump, allowing O. formigenes to display decarboxylative phosphorylation. The role played by OxlT argues that membrane carriers can assume unanticipated emergent properties when their biochemical functions are properly articulated in relation to other aspects of cell function.

Identification of a residue in the translocation pathway of a membrane carrier.

Preliminary work using directed mutagenesis proved that cysteine is not required for operation of UhpT, the anion exchange protein responsible for glucose 6-phosphate transport by E. coli. We then made a detailed study of C143 and C265, because these cysteines impart sensitivity to p-chloromercuribenzosulfonate (PCMBS), a sulfhydral agent resembling glucose 6-phosphate in size, shape, and charge. We showed that C143 was exposed to the cytoplasm, as expected from hydropathy analysis, but we found no sidedness for C265. Rather, C265 was accessible to PCMBS from both membrane surfaces. And since the attack at C265 was blocked by glucose 6-phosphate, position 265 must lie directly on the pathway taken by the substrate as it moves through this membrane carrier.

Coenzyme A-acylating aldehyde dehydrogenase from Clostridium beijerinckii NRRL B592.

Acetaldehyde and butyraldehyde are substrates for alcohol dehydrogenase in the production of ethanol and 1-butanol by solvent-producing clostridia. A coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH), which also converts acyl-CoA to aldehyde and CoA, has been purified under anaerobic conditions from Clostridium beijerinckii NRRL B592. The ALDH showed a native molecular weight (Mr) of 100,000 and a subunit Mr of 55,000, suggesting that ALDH is dimeric. Purified ALDH contained no alcohol dehydrogenase activity. Activities measured with acetaldehyde and butyraldehyde as alternative substrates were copurified, indicating that the same ALDH can catalyze the formation of both aldehydes for ethanol and butanol production. Based on the Km and Vmax values for acetyl-CoA and butyryl-CoA, ALDH was more effective for the production of butyraldehyde than for acetaldehyde. ALDH could use either NAD(H) or NADP(H) as the coenzyme, but the Km for NAD(H) was much lower than that for NADP(H). Kinetic data suggest a ping-pong mechanism for the reaction. ALDH was more stable in Tris buffer than in phosphate buffer. The apparent optimum pH was between 6.5 and 7 for the forward reaction (the physiological direction; aldehyde forming), and it was 9.5 or higher for the reverse reaction (acyl-CoA forming). The ratio of NAD(H)/NADP(H)-linked activities increased with decreasing pH. ALDH was O2 sensitive, but it could be protected against O2 inactivation by dithiothreitol. The O2-inactivated enzyme could be reactivated by incubating the enzyme with CoA in the presence or absence of dithiothreitol prior to assay.

Oxidation product(s) in acetaldehyde reacts with NAD(P)H and interferes with assay of alcohol dehydrogenase.

A decrease in absorbance at 340 nm, at rates similar to those obtained with alcohol dehydrogenases in routine assays, occurred when NADH or NADPH was mixed with acetaldehyde that had been exposed to air for various durations. NAD(P)H was apparently oxidized by interfering substance(s) present in acetaldehyde. Reagent-grade acetaldehyde from newly opened bottles as well as acetaldehyde redistilled under strictly O2-free conditions contained minimal amounts of NAD(P)H-reacting substance(s). Redistillation under poor anaerobic conditions or in air increased the amount of NAD(P)H-reacting substance(s) in redistilled acetaldehyde. NADPH reacted at a higher rate than NADH with the interfering substance(s) in Tris-Cl buffer at pH 7.5. Also, the reaction was faster in Tris buffer than in phosphate buffer at pH 7.5. The NAD(P)H-oxidizing reaction may not be apparent when the nominal concentration of acetaldehyde used was below 5 mM, but the measured ethanol dehydrogenase activity could be significantly lower with acetaldehyde containing a measurable level of interfering substance(s). This study suggests that acetaldehyde is most easily tested with NADPH for the presence of a significant level of interfering substance(s) and that redistillation, if necessary, must be performed under strictly O2-free conditions.

Butanol-Ethanol Dehydrogenase and Butanol-Ethanol-Isopropanol Dehydrogenase: Different Alcohol Dehydrogenases in Two Strains of Clostridium beijerinckii (Clostridium butylicum).

Alcohol-producing strains of Clostridium beijerinckii (Clostridium butylicum) produce, besides acetone, either n-butanol and ethanol or n-butanol, ethanol, and isopropanol as their characteristic products. Alcohol dehydrogenase has been isolated from a strain (NRRL B593) of C. beijerinckii producing isopropanol and from a strain (NRRL B592) not producing isopropanol. Butanol-ethanol dehydrogenase activities were present in both strains, but isopropanol dehydrogenase activity was present only in the isopropanol-producing strain. The butanol-ethanol dehydrogenase of strain NRRL B592 had M(r) 66,000 and a K(m) of 6 muM for butyraldehyde. In contrast, the butanol-ethanol-isopropanol dehydrogenase of strain NRRL B593 had a M(r) 100,000 and K(m)s of 9.5 and 1.0 mM for butyraldehyde and acetone, respectively. In a purification by four different types of separatory methods (DEAE-cellulose, hydroxyapatite, Sephacryl S-300, and Matrex Gel Red A), butanol-ethanol-isopropanol dehydrogenase activities of strain NRRL B593 were purified up to 200-fold (10 to 30% yield), and these activities were not separated. Gel electrophoresis followed by activity stain also revealed distinct mobilities for the butanol-ethanol dehydrogenase of strain NRRL B592 and the butanol-ethanol-isopropanol dehydrogenase of strain NRRL B593. In cell extracts from both strains, a higher alcohol dehydrogenase activity was measured with NADP(H) than with NAD(H). The 150- to 200-fold-purified alcohol dehydrogenase from strain NRRL B593 did not show any NAD(H)-linked activities. The K(m) for NADPH was 31 muM (with butyraldehyde as cosubstrate) and 18 muM (with acetone as cosubstrate) for the alcohol dehydrogenase of strain NRRL B593. This study showed that the alcohol dehydrogenases from two strains of C. beijerinckii differed significantly.