Advanced crystallisation methods for small organic molecules.

Molecular materials based on small organic molecules often require advanced structural analysis, beyond the capability of spectroscopic techniques, to fully characterise them. In such cases, diffraction methods such as single crystal X-ray diffraction (SCXRD), are one of the most powerful tools available to researchers, providing molecular and structural elucidation at atomic level resolution, including absolute stereochemistry. However SCXRD, and related diffraction methods, are heavily dependent on the availability of suitable, high-quality crystals, thus crystallisation often becomes the major bottleneck in preparing samples. Following a summary of classical methods for the crystallisation of small organic molecules, this review will focus on a number of recently developed advanced methods for crystalline material sample preparation for SCXRD. This review will cover two main areas of modern small organic molecule crystallisation, namely the inclusion of molecules within host complexes (e.g., "crystalline sponge" and tetraaryladamantane based inclusion chaperones) and the use of high-throughput crystallisation, employing "under-oil" approaches (e.g., microbatch under-oil and ENaCt). Representative examples have been included for each technique, together with a discussion of their relative advantages and limitations to aid the reader in selecting the most appropriate technique to overcome a specific analytical challenge.

Identification of casein kinase I substrates by in vitro expression cloning screening.

Casein kinase I (CKI) is a widely expressed protein kinase family implicated in diverse processes including membrane trafficking, DNA repair, and circadian rhythm. Despite the large number of CKI genes, few biologically relevant substrates have been identified. As an approach to better defining the spectrum of CKI substrates, we extended a recently described in vitro expression cloning (IVEC) strategy. Polypeptides pools were screened for kinase-dependent electrophoretic mobility shifts. Ten putative CKI substrates were isolated from an initial sample of 3000 random cDNA clones. Candidate substrates include proteins involved in RNA metabolism (a putative RNA helicase, the nucleolar protein hNOP56, and hnRNP A1, and ribosomal proteins L4, L8, and L13), as well as keratin 17, a necdin-related protein, and the calcium-binding proteins desmoglein 2 and annexin II. The same pools were also screened with active ERK2, and four substrates identified: aldolase, NSD-like protein, uracil-DNA glycosylase, and HHR23A. IVEC is an effective method to identify novel protein kinase substrates.

Hemizygosity for the COP9 signalosome subunit gene, SGN3, in the Smith-Magenis syndrome.

Smith-Magenis syndrome (SMS) is a multiple congenital anomaly/mental retardation syndrome associated with an interstitial deletion of chromosome band 17p11.2. The critical region is extremely gene-rich and spans approximately 1.5-2.0 Mb of DNA. Here we report the localization and partial characterization of the gene for subunit 3 of the COP9 signalosome, SGN3. SGN3 maps to the distal portion of the SMS critical interval, between SREBF1 and cCI17-638. We assessed the potential effect of haploinsufficiency of SGN3 in SMS patient lymphoblastoid cell lines through transfection studies and western analysis. Our results indicate that the COP9 signalosome assembles properly in these cells and appears to have normal expression and a kinase function intact. However, because the role of the COP9 signalosome in embryogenesis or differentiation is still uncertain, we cannot rule out the involvement of this gene in the Smith-Magenis syndrome.

Biochemical variants of Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz (SLO or RSH) syndrome is characterized by multiple congenital anomalies, mental retardation, and defective growth; it results from an inherited defect in the biosynthesis of cholesterol. Patients have elevated plasma concentrations of 7-dehydrocholesterol, the immediate biosynthetic precursor of cholesterol and most also have low circulating levels of cholesterol. To understand better the biochemical basis of clinical variability, we evaluated cholesterol biosynthesis in lymphoblasts from 3 unrelated SLOS patients with distinct phenotypes. One patient has "type I SLOS", the second has the more severe "type II SLOS" and the third is classified as atypical and had been postulated to have a defect in sterol transport. The lymphoblasts of each patient show normal subcellular localization of cholesterol and 7-dehydrocholesterol by gradient fractionation. Biochemical differences in the ability of the lymphoblasts to convert 7-dehydrocholesterol to cholesterol are described and correspond to the severity of disease (type II > type I > atypical). Recently, the gene responsible for most SLOS cases (DHCR7) was mapped to chromosome 11 and mutations in DHCR7 were found in each of these patients. The biochemical differences described here likely result from the different mutations observed in DHCR7.

Acute and long-term effects of the angiotensin-converting enzyme inhibitor, enalapril, on adrenergic activity and sensitivity during exercise in patients with left ventricular systolic dysfunction.

Patients with heart failure and left ventricular systolic dysfunction exhibit increased adrenergic activity but blunted adrenergic responsiveness. We studied patients enrolled in the Studies of Left Ventricular Dysfunction, examining exercise responses of heart rate (HR) and plasma norepinephrine (PNE). Eighty-seven patients were studied before randomization; 65 of these were examined 1 year after randomization to placebo or enalapril. Compared with prevention trial (asymptomatic) patients, patients in the treatment trial (symptomatic) had higher resting HR and PNE levels and less increase in HR with a greater increase in PNE with exercise. Acute administration of enalapril increased the resting HR in patients in the prevention trial only but had no significant effect on PNE. After 1 year of therapy, patients in the prevention trial exhibited no change. Within the treatment trial, the placebo group displayed both a higher peak PNE and increase in PNE with exercise than did the enalapril group, whose HR response was maintained in spite of a reduction of exercise PNE. We conclude that (1) compared with asymptomatic patients, symptomatic patients with reduced left ventricular ejection fraction manifest greater resting and exercise adrenergic activity, with blunted HR response; and (2) in symptomatic patients, 1 year of enalapril treatment effected an augmented HR response to adrenergic stimulation, supporting an interaction between the renin/angiotensin and adrenergic nervous systems. Normalization of adrenergic tone and response likely contributes to the benefits of long-term angiotensin-converting enzyme inhibitor therapy.

Role of multidrug resistance P-glycoproteins in cholesterol esterification.

Cholesterol esterification, catalyzed by acyl-CoA:cholesterol acyltransferase (ACAT), plays a central role in cellular cholesterol homeostasis and in physiologic processes that lead to coronary heart disease. Although ACAT resides in the endoplasmic reticulum (ER), the cholesterol substrate for esterification originates in the plasma membrane and must be transported to the ER for esterification. Progesterone inhibits esterification, possibly by blocking the transport of cholesterol to the ER. Recent studies suggest that progesterone acts by inhibiting the activity of one or more of the multidrug-resistant (MDR) P-glycoproteins. In the current manuscript, we demonstrate that progesterone's ability to inhibit esterification is not mediated through the progesterone receptor. We evaluate a series of steroid hormones and find a strong correlation between a steroid hormone's hydrophobicity and its ability to inhibit both cholesterol esterification and MDR-catalyzed drug efflux. We also find that cholesterol esterification is inhibited by nonsteroidal MDR inhibitors, and that this inhibition specifically affects the esterification of cholesterol derived from the plasma membrane. MDR inhibitors also inhibit cholesterol esterification in a wide range of cultured human cell lines. These observations suggest that MDR activity normally functions in a general process of intracellular cholesterol transport.

Role for sterol regulatory element binding protein in the regulation of farnesyl diphosphate synthase and in the control of cellular levels of cholesterol and triglyceride: evidence from sterol regulation-defective cells.

In order to define the factors involved in the regulation of farnesyl diphosphate (FPP) synthase, we used sterol regulation-defective (SRD) cell lines that constitutively express either high (SRD-2) or low (SRD-6) levels of transcriptionally active sterol regulatory element binding protein (SREBP). FPP synthase mRNA levels were high in SRD-2 cells and low in SRD-6 cells and were unaffected by the addition or removal of sterols from the media. In contrast, the mRNA levels in parental CHO-7 cells were regulated by sterols. SRD-2, SRD-6, and CHO-7 cells were also transiently transfected with plasmids containing FPP synthase promoter-reporter genes. Reporter gene activity was significantly higher in SRD-2 cells than in either SRD-6 or CHO-7 cells, consistent with a higher rate of transcription of the reporter gene in SRD-2 cells. The high expression of the reporter gene in SRD-2 cells was not observed when the FPP synthase promoter contained a three base pair mutation within an SREBP binding site, termed sterol regulatory element-3 (SRE-3). These observations are consistent with the hypothesis that high levels of transcription of the FPP synthase gene are dependent on the availability of transcriptionally active SREBP. We also demonstrate that the incorporation of radioactive acetate into both cholesterol and fatty acids was enhanced in SRD-2 cells as compared to CHO-7 or SRD-6 cells. Finally, we demonstrate that the concentrations of cholesterol, cholesteryl ester, and triglyceride were all significantly elevated in SRD-2 cells. We conclude that SREBP is involved not only in the regulation of FPP synthase and cholesterogenesis but also in fatty acid and triglyceride synthesis.

Progesterone inhibits cholesterol biosynthesis in cultured cells. Accumulation of cholesterol precursors.

Cells acquire cholesterol through endogenous synthesis and through receptor-mediated uptake of cholesterol-rich low density lipoprotein (LDL). Esterification of LDL-derived cholesterol is catalyzed by acyl-CoA:cholesterol acyltransferase (ACAT) in the endoplasmic reticulum (ER). Progesterone inhibits esterification, and, although the mechanism of inhibition is not completely understood, this inhibition results from progesterone's ability to inhibit the activity of multiple drug resistance (MDR) P-glycoproteins (P. DeBry and J. E. Metherall, submitted for publication). In the current manuscript, we demonstrate that progesterone inhibits cholesterol biosynthesis resulting in the accumulation of a number of sterol precursors. In Chinese hamster ovary (CHO) cells, high concentrations (100 microM) of progesterone completely blocked cholesterol production, resulting in the accumulation of lanosterol and a lanosterol precursor. Lower concentrations (40 microM) of progesterone cause plasma membrane accumulation of several sterol products. The majority of these sterols are precursors of cholesterol since they were efficiently converted to cholesterol upon removal of progesterone from the culture medium. Although very high concentrations (> 200 microM) of progesterone killed CHO cells, their growth was restored by the addition of cholesterol to the growth medium, indicating that progesterone toxicity resulted from cholesterol auxotrophy. The effect of progesterone was not unique to CHO cells; progesterone also inhibited cholesterol biosynthesis in all human cell lines tested. These observations suggest that a common progesterone-sensitive pathway is involved in both cholesterol biosynthesis and the processing of LDL-derived cholesterol.

Role of multidrug resistance P-glycoproteins in cholesterol biosynthesis.

Multidrug resistance (MDR) P-glycoproteins were first recognized for their ability to catalyze ATP-dependent efflux of cytotoxic agents from tumor cells when overexpressed. Despite extensive study, little is known about the normal substrate(s) and normal cellular function of these proteins. In the accompanying manuscript (Metherall, J. E., Waugh, K., and Li, H. (1996) J. Biol. Chem. 271, 2627-2633), we demonstrate that progesterone inhibits cholesterol biosynthesis, causing the accumulation of a number of cholesterol precursors. In the current manuscript, we use several criteria to show that the progesterone receptor is not involved in this inhibition. Rather, we demonstrate that progesterone inhibits cholesterol biosynthesis by interfering with MDR activity. We show that a steroid hormone's ability to inhibit cholesterol biosynthesis is correlated with: 1) its general hydrophobicity and 2) its ability to inhibit MDR activity. The only exception to this finding is beta-estradiol, which is a more potent inhibitor of cholesterol biosynthesis than expected based solely on hydrophobicity and MDR inhibition. We further demonstrate that nonsteroidal inhibitors of MDR also inhibit cholesterol biosynthesis. Since MDR activity is required for esterification of LDL-derived cholesterol (P. DeBry and J. E. Metherall, submitted for publication), we investigated the relationship between these phenomena and show that inhibition of cholesterol esterification does not cause inhibition of cholesterol biosynthesis and that inhibition of cholesterol biosynthesis does not cause inhibition of cholesterol esterification. We propose a model in which MDR is required for transport of sterols from the plasma membrane to the endoplasmic reticulum (ER). Inhibiting this transport prevents cholesterol esterification and cholesterol biosynthesis by preventing sterol substrates from reaching ER-resident enzymes.

Enhanced hepatic uptake of thallium-201 in patients with severe narrowing of the right coronary artery.

To assess the hepatic uptake of thallium-201 after exercise treadmill testing and to investigate whether hepatic uptake of thallium-201 may be a useful marker of right coronary artery (RCA) disease, 43 patients were studied: 17 with RCA disease (9 with 1-vessel disease, 8 with multivessel disease including the RCA), 8 with left coronary system disease alone, and 18 with a low probability (< 5%) of coronary disease. All subjects were studied with symptom-limited exercise and redistribution thallium-201 single-photon emission computed tomographic (SPECT) scintigraphy. Two indexes of hepatic uptake were derived: a liver-to-heart ratio after stress, and a stress-to-rest hepatic ratio. The low-probability group had a liver/heart ratio of 0.48 +/- 0.02. In the group with RCA disease alone, liver/heart ratio was 1.29 +/- 0.20 (p < 0.005 vs low-probability group). Patients with multivessel coronary artery disease involving the RCA had a ratio of 1.19 +/- 0.16 (p < 0.005 vs low-probability group), and patients with only left coronary system disease had a liver/heart ratio of 0.87 +/- 0.15 (p < 0.05 vs low-probability group). The stress/rest ratio of the low-probability group was 0.83 +/- 0.04. Patients with RCA disease alone had a stress/rest ratio of 1.49 +/- 0.25 (p < 0.05 vs low-probability group), and patients with multivessel disease involving the RCA had a stress/rest ratio of 1.16 +/- 0.08 (p < 0.005 vs low-probability group).(ABSTRACT TRUNCATED AT 250 WORDS)

Predicting recovery of severe regional ventricular dysfunction. Comparison of resting scintigraphy with 201Tl and 99mTc-sestamibi.

BACKGROUND: Regional 201Tl activity after resting injection, imaged early and after redistribution, reflects viable myocardium and can predict improved isotope uptake as well as regional and global ventricular function after revascularization. 99mTc-sestamibi, a perfusion tracer with favorable imaging characteristics, has distinct kinetics compared with 201Tl, demonstrating minimal redistribution; this property may give 201Tl an advantage for detecting viable myocardium, particularly in segments with resting hypoperfusion. The purpose of this study was to compare regional activities of 201Tl and 99mTc-sestamibi after resting injections in patients with coronary artery disease and regional or global left ventricular dysfunction and to assess their comparative abilities for predicting recovery of severe regional ventricular dysfunction after revascularization. METHODS AND RESULTS: Qualitative and quantitative comparisons of rest and redistribution 201Tl activity and sestamibi activity 1 hour after rest injection were performed in 31 patients with coronary artery disease and left ventricular dysfunction. Quantitative analysis of three short-axis tomograms per patient was performed by use of circumferential profiles that allowed analysis of 12 segments per patient. Two-dimensional echocardiography was used to assess wall motion and thickening in segments corresponding to the single photon emission computed tomography data. Concordance between regional 201Tl activity at redistribution imaging and regional sestamibi activity by semiquantitative visual analysis demonstrated concordant regional activity in 87% of segments; among discordant segments, no significant skew was seen, indicating enhanced uptake of one agent over the other. Quantitative analysis for all segments showed significant correlation (r = .86, P < .001) between quantitative regional 201Tl redistribution activity and 1-hour post-rest injection sestamibi activity in individual segments. Eighteen of these patients were revascularized, and echocardiography was repeated 20 +/- 16 days later; segments exhibiting significant regional ventricular dysfunction before revascularization were classified as having reversible or irreversible dysfunction on the basis of the change in wall motion and thickening. 201Tl and sestamibi regional activities were similar in those segments with reversible (72 +/- 11% [percent of peak activity] versus 75 +/- 9%, respectively, P = NS) as well as irreversible ventricular dysfunction (51 +/- 11% versus 50 +/- 8%, P = NS). Positive (75% versus 80% for 201Tl and sestamibi, respectively) and negative (92% versus 96%, respectively) predictive values for recovery of regional ventricular dysfunction after revascularization were similar for the two agents. CONCLUSIONS: In patients with coronary artery disease and left ventricular dysfunction, quantified sestamibi activity 1 hour after rest injection parallels redistribution 201Tl activity after a resting injection, suggesting that uptake and subsequent handling of sestamibi are more complex than can be explained by a pure flow tracer with no redistribution. Quantitative analysis of regional activities of both 201Tl and sestamibi after resting injections can differentiate viable from nonviable myocardium, and the two agents comparably predict reversibility of significant regional wall motion abnormalities after revascularization in such patients to a similar degree.

Loss of transcriptional activation of three sterol-regulated genes in mutant hamster cells.

Cholesterol biosynthesis and uptake are controlled by a classic end product-feedback mechanism whereby elevated cellular sterol levels suppress transcription of the genes encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and the low-density lipoprotein receptor. The 5'-flanking region of each gene contains a common cis-acting element, designated the sterol regulatory element (SRE), that is required for transcriptional regulation. In this report, we describe mutant Chinese hamster ovary (CHO) cell lines that lack SRE-dependent transcription. Mutant cell lines were isolated on the basis of their ability to survive treatment with amphotericin B, a polyene antibiotic that kills cells by interacting with cholesterol in the plasma membrane. Four mutant lines (SRD-6A, -B, -C, and -D) were found to be cholesterol auxotrophs and demonstrated constitutively low levels of mRNA for all three sterol-regulated genes even under conditions of sterol deprivation. The mutant cell lines were found to be genetically recessive, and all four lines belonged to the same complementation group. When transfected with a plasmid containing a sterol-regulated promoter fused to a bacterial reporter gene, SRD-6B cells demonstrated constitutively low levels of transcription, in contrast to wild-type CHO cells, which increased transcription under conditions of sterol deprivation. Mutation of the SREs in this plasmid prior to transfection reduced the level of expression in wild-type CHO cells deprived of sterols to the level of expression found in SRD-6B cells. The defect in SRD-6 cells is limited to transcriptional regulation, since posttranscriptional mechanisms of sterol-mediated regulation were intact: the cells retained the ability to posttranscriptionally suppress HMG-CoA reductase activity and to stimulate acyl-CoA:cholesterol acyltransferase activity. These results suggest that SRD-6 cells lack a factor required for SRE-dependent transcriptional activation. We contrast these cells with a previously isolated oxysterol-resistant cell line (SRD-2) that lacks a factor required for SRE-dependent transcriptional suppression and propose a model for the role of these genetically defined factors in sterol-mediated transcriptional regulation.

Effectiveness of preload reserve as a determinant of clinical status in patients with left ventricular systolic dysfunction. The SOLVD Investigators.

The hemodynamic determinants of clinical status in patients with left ventricular (LV) systolic dysfunction have not been established. In the present study, preload reserve--LV distension during exercise--was related to clinical status, and the effect of acute angiotensin-converting enzyme inhibition was examined in 97 patients with ejection fraction less than or equal to 0.35 enrolled in the trial, Studies of Left Ventricular Dysfunction (SOLVD). Sixty-one asymptomatic patients (group I) were compared with 36 patients with symptomatic heart failure (group II). Radionuclide LV volumes were measured at rest and during maximal cycle exercise. Group II patients had higher resting heart rates, end-diastolic and end-systolic volumes, and lower ejection fractions (all p less than 0.005). During exercise, only patients in group I had increased stroke volume (from 35 +/- 8 to 39 +/- 11 ml/m2 [mean +/- SD; p less than 0.0005]) due to an increase in end-diastolic volume (from 119 +/- 29 to 126 +/- 29 ml/m2 [p less than 0.0005]), contributing to a greater increase in LV minute output (p less than 0.0001, group I vs group II). After administration of intravenous enalapril (1.25 mg), LV end-diastolic volume response to exercise was augmented in group II (rest, 140 +/- 42; exercise, 148 +/- 43 ml/m2; p less than 0.0005) and LV output response increased slightly (p less than 0.05). Thus, in patients with asymptomatic systolic dysfunction, recruitment of preload during exercise is responsible for maintaining a stroke volume contribution to the cardiac output response.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression cloning of a diphtheria toxin receptor: identity with a heparin-binding EGF-like growth factor precursor.

A monkey cDNA (pDTS) encoding a diphtheria toxin (DT) sensitivity determinant was isolated by expression cloning in mouse L-M cells. Mouse cells are naturally resistant to DT, because they lack functional cell surface receptors for the toxin. Unlike wild-type L-M cells, pDTS-transfected mouse cells are extremely toxin sensitive and specifically bind radioiodinated DT. Intoxication of the transfected cells requires receptor-mediated endocytosis of the bound toxin. The cDNA is predicted to encode an integral membrane protein that is identical to the precursor of a heparin-binding EGF-like growth factor. The DT sensitivity protein is thus a growth factor precursor that DT exploits as a receptor.

A 25-hydroxycholesterol-resistant cell line deficient in acyl-CoA: cholesterol acyltransferase.

We describe a line of mutant Chinese hamster ovary cells, SRD-4 cells, that lacks acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity and fails to synthesize cholesteryl esters when stimulated with 25-hydroxycholesterol or low density lipoprotein. The cells also have a partial defect in their ability to repress transcription of three sterol-regulated genes, 3-hydroxy-3-methylglutaryl-coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and the low density lipoprotein receptor. The cells were selected by mutagenesis followed by growth in the presence of 25-hydroxycholesterol, which kills the parental cells by cholesterol depletion, owing to an inhibition of cholesterol synthesis and a stimulation of cholesterol esterification. Treatment of parental cells with compound 58-035 (3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1- phenylethyl]propanamide), an inhibitor of ACAT, abolished cholesterol esterification but did not reproduce the defect in gene repression seen in the SRD-4 cells, and it only partially reproduced the resistance to the killing effect of 25-hydroxycholesterol. We conclude that the SRD-4 cells most likely have two independent defects, one in ACAT and the other in a factor that mediates sterol-dependent transcriptional repression. The SRD-4 cells thus resemble a line of hamster cells previously isolated (Cadigan, K.M., Heider, J.G., and Chang, T.-Y. (1988) J. Biol. Chem. 263, 274-282), which has similar independent defects. The results raise the possibility that a partial resistance to sterol repression provides a growth advantage to cells that lack ACAT.

Genetic distinction between sterol-mediated transcriptional and posttranscriptional control of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Sterols reduce the activity of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMG-CoA reductase) transcriptionally by inhibiting the synthesis of reductase mRNA and posttranscriptionally by accelerating degradation of the enzyme. We and others have described mutant lines of Chinese hamster fibroblasts that are completely resistant to sterol-mediated repression of transcription of HMG-CoA reductase as well as two other sterol-regulated genes, HMG-CoA synthase and the low density lipoprotein (LDL) receptor. In the current studies, we show that one line of sterol-resistant mutant cells (SRD-3 cells) retains the ability to slow the degradation of HMG-CoA reductase by 7-fold in response to treatment with compactin, an inhibitor of reductase that blocks sterol synthesis. The compactin effect is reversed by exogenous sterols. Similar results were obtained with another mutant line of sterol-resistant cells (SRD-2 cells) whose defective transcriptional regulation is attributable to a different gene than that in the SRD-3 cells, as determined by complementation analysis. These data indicate that the gene products that are defective in the SRD-3 and SRD-2 cells are not required for the sterol-mediated regulation of degradation of HMG-CoA reductase. Thus, mammalian cells possess at least two genetically distinct mechanisms, one transcriptional and the other posttranscriptional, for sensing and responding to the intracellular level of sterols.

Effect of acute angiotensin converting enzyme inhibition on left ventricular filling in patients with congestive heart failure. Relation to right ventricular volumes.

To examine the manner in which changes in diastolic performance can contribute to the effect of vasodilation in patients with left ventricular (LV) systolic dysfunction, we examined the effect of acute inhibition of angiotensin converting enzyme with intravenous enalaprilat on early LV diastolic filling. We studied 43 patients with congestive heart failure and depressed LV systolic function (mean ejection fraction +/- SD, 0.24 +/- 0.06), performing radionuclide ventriculography before and after administration of 1.25 mg intravenous enalaprilat. We measured the effect of enalaprilat on the maximum rate of early LV diastolic filling normalized in four different ways and related these changes to both LV and right ventricular (RV) volumes. Enalaprilat induced a small but statistically significant reduction in LV end-systolic volume and increase in LV ejection fraction. For the entire patient group, there was no significant change in LV peak filling rate after enalaprilat administration. For individual patients, however, the effect of enalaprilat on peak filling rate was related to resting RV end-diastolic and end-systolic volumes. In patients with enlarged RV end-diastolic volumes (greater than or equal to 120 ml/m2), mean peak filling rate increased from 1.38 +/- 0.6 to 1.71 +/- 0.6 end-diastolic volumes (EDV)/sec and from 244 +/- 131 to 297 +/- 162 ml/sec/m2 after enalaprilat administration, whereas no change in mean peak filling rate was observed in patients with nondilated RVs. These observations were present regardless of the method of normalizing peak filling rate. Thus, the response of LV peak filling rate to enalaprilat is influenced by the presence of RV dilatation.(ABSTRACT TRUNCATED AT 250 WORDS)

Loss of transcriptional repression of three sterol-regulated genes in mutant hamster cells.

Two genes that encode enzymes in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and HMG-CoA synthase, and the gene encoding the low density lipoprotein (LDL) receptor are repressed when sterols accumulate in animal cells. Their 5'-flanking regions contain a common element, designated sterol regulatory element-1 (SRE-1). In the HMG-CoA synthase and LDL receptor promoters, the SRE-1 enhances transcription in the absence of sterols and is inactivated in the presence of sterols. In the HMG-CoA reductase promoter, the region containing the SRE-1 represses transcription when sterols are present. In the current studies, we show that the SRE-1 retains enhancer function but loses sterol sensitivity in mutant Chinese hamster ovary cells that are resistant to the repressor, 25-hydroxycholesterol. In the absence of sterols, the mutant cells produced high levels of all three sterol-regulated mRNAs, and there was no repression by 25-hydroxycholesterol. When transfected with plasmids containing each of the regulated promoters fused to a bacterial reporter gene, the mutant cells showed high levels of transcription in the absence of sterols and no significant repression by sterols. When the SRE-1 in the LDL receptor and HMG-CoA synthase promoters was mutated prior to transfection into the mutant cells, transcription was markedly reduced. Thus, the 25-hydroxycholesterol-resistant cells retain a protein that enhances transcription by binding to the SRE-1 in the absence of sterols, but they have lost the function of a protein that abolishes this enhancement in the presence of sterols. Mutation of a 30-base pair segment of the HMG-CoA reductase promoter that contains the SRE-1 did not reduce transcription in the mutant cells, indicating that this promoter is driven by elements other than the SRE-1. Nevertheless, this promoter failed to be repressed by sterols in the mutant cells. These data suggest that a common factor mediates the effects of sterols on the SRE-1 in all three promoters and that this factor has been functionally lost in the 25-hydroxycholesterol-resistant cells.

Dipyridamole thallium-201 imaging pre- and post-coronary angioplasty for assessment of regional myocardial ischemia in humans.

To examine the effect of coronary angioplasty on myocardial perfusion and to examine anatomic correlates of dipyridamole-thallium images, we performed dipyridamole thallium scanning before and after 24 angioplasty procedures in 23 patients with clinical ischemia. Dipyridamole thallium tomography was performed 1 to 4 (mean +/- S.D. 1.5 +/- 0.9) days before and 1 to 25 (6.3 +/- 6.9) days after angioplasty. Coronary angioplasty was anatomically successful (less than 50% residual stenosis) in 23 of 24 patients. Before angioplasty, 3 of 24 scans were interpreted as normal, with no change in these patients following angioplasty. Prior to angioplasty, 19 scans showed redistribution. After successful coronary angioplasty in 18 of 19, 17 showed improvement in dipyridamole thallium scanning; one did not show improvement and later required repeat coronary dilatation. In one patient with failed angioplasty, images before and after angioplasty were unchanged. Nine scans showed "fixed" defects before angioplasty, with improvement in three of nine following angioplasty. Quantitative analysis of the dipyridamole thallium scans revealed significant improvement in uptake in the myocardial regions supplied by the successfully dilated artery (involved regions), but not in the remote (uninvolved) regions. We conclude that myocardial perfusion, as judged by dipyridamole thallium scanning, almost always improves following anatomically successful coronary angioplasty. "Fixed" defects may improve following angioplasty and presumably represent ischemia with incomplete redistribution, rather than infarction.