Biomedical subjects
G Brooks
Publications and source records attributed to G Brooks.
Arterial disease and thrombosis in the antiphospholipid syndrome: a pathogenic role for endothelin 1.
OBJECTIVE: To explore a possible correlation between endothelin 1 (ET-1), the most potent endothelium-derived contracting factor that modulates vascular smooth muscle tone, and arterial disease in patients with the antiphospholipid syndrome (APS). METHODS: Plasma levels of ET-1 were measured in APS patients with (n = 16) and without (n = 11) arterial thrombosis and in non-APS patients with arterial thrombosis (n = 9). In addition, steady-state prepro-ET-1 messenger RNA (mRNA) levels were determined in endothelial cells treated with a range of human monoclonal anticardiolipin antibodies (aCL) (as anti-beta2-glycoprotein I antibodies) by semiquantitative 32P-dCTP-labeled reverse transcription-polymerase chain reaction. RESULTS: Compared with healthy controls, markedly increased plasma levels of ET-1 were found in APS patients with arterial thrombosis (2.00 +/- 0.87 versus 0.96 +/- 0.37 pg/ml; P = 0.0001) but not in other groups. Three human monoclonal aCL induced prepro-ET-1 mRNA levels significantly more than did control monoclonal antibody lacking aCL activity. CONCLUSION: Plasma ET-1 levels correlated significantly with a history of arterial thrombosis in patients with APS. Prepro-ET-1 mRNA was induced by human monoclonal aCL in the in vitro experimental system. The induction of ET-1 by antiphospholipid antibodies might contribute to increased arterial tone, leading to vasospasm and, ultimately, to arterial occlusion.
Expressions and activities of cell cycle regulatory molecules during the transition from myocyte hyperplasia to hypertrophy.
The role of cell cycle dependent molecules in controlling the switch from cardiac myocyte hyperplasia to hypertrophy remains unclear, although in the rat this process occurs between day 3 and 4 after birth. In this study we have determined (1) cell cycle profiles by fluorescence activated cell sorting (FACS); and (2) expressions, co-expressions and activities of a number of cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and in vitro kinase assays in freshly isolated rat cardiac myocytes obtained from 2, 3, 4 and 5-day-old animals. The percentage of myocytes found in the S phase of the cell cycle decreased significantly during the transition from hyperplasia to hypertrophy (5.5, 3.5, 2.3 and 1.9% of cells in 2-, 3-, 4- and 5-day-old myocytes, respectively,P<0.05), concomitant with a significant increase in the percentage of G0/G1 phase cells. At the molecular level, the expressions and activities of G1/S and G2/M phase acting cyclins and CDKs were downregulated significantly during the transition from hyperplasia to hypertrophy, whereas the expressions and activities of G1 phase acting cyclins and CDKs were upregulated significantly during this transition. In addition, p21(CIP1)- and p27(KIP1)- associated CDK kinase activities remained relatively constant when histone H1 was used as a substrate, whereas phosphorylation of the retinoblastoma protein was upregulated significantly during the transition from hyperplasia to hypertrophy. Thus, there is a progressive and significant G0/G1 phase blockade during the transition from myocyte hyperplasia to hypertrophy. Whilst CDK2 and cdc2 may be pivotal in the withdrawal of cardiac myocytes from the cell cycle, CDK4 and CDK6 may be critical for maintaining hypertrophic growth of the myocyte during development.
Arresting developments in the cardiac myocyte cell cycle: role of cyclin-dependent kinase inhibitors.
Like most other cells in the body, foetal and neonatal cardiac myocytes are able to divide and proliferate. However, the ability of these cells to undergo cell division decreases progressively during development such that adult myocytes are unable to divide. A major problem arising from this inability of adult cardiac myocytes to proliferate is that the mature heart is unable to regenerate new myocardial tissue following severe injury, e.g. infarction, which can lead to compromised cardiac pump function and even death. Studies in proliferating cells have identified a group of genes and proteins that controls cell division. These proteins include cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors (CDKIs), which interact with each other to form complexes that are essential for controlling normal cell cycle progression. A variety of other proteins, e.g. the retinoblastoma protein (pRb) and members of the E2F family of transcription factors, also can interact with, and modulate the activities of, these complexes. Despite the major role that these proteins play in other cell types, little was known until recently about their existence and activities in immature (proliferating) or mature (non-proliferating) cardiac myocytes. The reason(s) why cardiac myocytes lose their ability to divide during development remains unknown, but if strategies were developed to understand the mechanisms underlying cardiac myocyte growth, it could open up new avenues for the treatment of cardiovascular disease. In this article, we shall review the function of the cell cycle machinery and outline some of our recent findings pertaining to the involvement of the cell cycle in modulating cardiac myocyte growth and hypertrophy.
The efficacy and safety of salmeterol compared to theophylline: meta-analysis of nine controlled studies.
The aim of this study was to compare the efficacy and safety of salmeterol vs theophylline in asthma management using meta-analysis of clinical trials. Nine clinical studies, containing a total of 1330 patients, met meta-analysis inclusion criteria: randomized, controlled study, minimum 2-week treatment duration with either salmeterol or theophylline. The main outcome measurements were morning and evening peak expiratory flow rate (PEFR), morning and evening symptom scores, use of salbutamol as rescue medication, and withdrawal from treatment for any cause. During the second week of treatment, salmeterol patients had a 10 l min-1 greater increase in mean morning PEFR from baseline than theophylline patients (P < 0.001). Similarly, in the second week, the increase in mean evening PEFR from baseline observed with salmeterol was significantly greater (P < 0.01) than that observed with theophylline. Salmeterol also produced a significantly greater increase in mean morning and evening PEFR than theophylline at weeks 3 and 4. Patients receiving salmeterol were free from daytime symptoms for a mean of 51% of days in the second week compared to 39% for theophylline patients (P < 0.001). Salmeterol patients experienced a mean of 63% symptom-free nights compared to 52% for theophylline patients (P < 0.001). Rescue medication with salbutamol was not required on 49% of days for salmeterol patients and 34% of days for theophylline patients. All results were maintained in the third and fourth weeks of treatment. Withdrawal and incidence of adverse events leading to withdrawal were significantly less frequent in patients receiving salmeterol (P < 0.001). Thus, this meta-analysis suggests that salmeterol has a superior safety and efficacy profile to theophylline in the management of symptoms of chronic asthma.
Role of G1 phase cyclins and cyclin-dependent kinases during cardiomyocyte hypertrophic growth in rats.
Cell cycle regulatory molecules are implicated in cardiomyocyte hypertrophy. We have investigated protein expression of cyclins A, D1-3, and E and cyclin-dependent kinases (CDKs) 2, 4, 5, and 6 in left ventricular (LV) tissues during the development of LV hypertrophy in rats following aortic constriction (AC). Compared with their expression in sham-operated controls (SH), expression of cyclins D2 and D3 and of CDK4 and CDK6 increased significantly from day 3 to day 21 after AC concomitant with increased LV mass. However, no significant difference was observed for CDK2 or CDK5. Cyclins A, D1, and E were undetectable. In vitro kinase activities of CDK4 and CDK6 increased approximately 70% from day 7 to day 14 in AC myocytes compared with SH myocytes (P < 0.03). Fluorescence-activated cell sorter analysis revealed a G0/G1 to G2/M phase progression in AC myocyte nuclei (22.0 +/- 1.1% in G2/M) by day 7 postoperation compared with progression in SH myocyte nuclei (14.0 +/- 0.8% in G2/M; P < 0.01). Thus an upregulation of certain cell cycle regulators is associated with cardiomyocyte hypertrophy.
Can shoulder dystocia be predicted? Preconceptive and prenatal factors.
OBJECTIVE: To evaluate the predictability of shoulder dystocia using preconceptive and prenatal risk factors. STUDY DESIGN: Data from 1,622 term patients with prenatal care prior to 20 weeks who delivered single, vertex fetuses during a consecutive 12-month period were analyzed. Two groups were chosen. The first group was patients whose fetuses experienced shoulder dystocia during delivery (cases). The second group (controls) consisted of the remaining patients, whose fetuses had not experienced shoulder dystocia. The two groups were compared with regard to demographics and pregnancy characteristics. RESULTS: Factors not significantly different between the two groups included were obesity, multiparity, history of diabetes, short maternal stature, postdatism and advanced maternal age. The incidence of macrosomia was significantly higher (P < .001) in cases (35.4%) than in controls (4.8%). Other factors associated with shoulder dystocia were previous shoulder dystocia, concurrent diabetes, prior delivery of a fetus > 4,000 g and excessive weight gain during pregnancy. Many factors previously associated with shoulder dystocia were found to be nonsignificant in our study. CONCLUSION: Macrosomia appears to be the single important factor associated with shoulder dystocia which, even in the presence of significant risk factors, remains largely unpredictable.
What clinical information do doctors need? Electronic medical references are being used by practitioners.
Explore the source record for details and available documents.
Cardiac Na+-H+ exchanger during postnatal development in the rat: changes in mRNA expression and sarcolemmal activity.
We examined Na+-H+ exchanger isoform 1 (NHE-1) mRNA expression in ventricular myocardium and its correlation with sarcolemmal NHE activity in isolated ventricular myocytes, during postnatal development in the rat. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA did not change in ventricular myocardium between 2 and 42 days after birth. Therefore, at seven time points within that age range. GAPDH expression was used to normalize NHE-1 mRNA levels, as determined by reverse transcription polymerase chain reaction analysis. There was a progressive five-fold reduction in NHE-1 mRNA expression in ventricular myocardium from 2 days to 42 days of age. As an index of NHE activity, acid efflux rates (J(H)) were determined in single neonatal (2-4-day-old) and adult (42-day-old) ventricular myocytes (n=16/group) loaded with the pH fluoroprobe carboxy-seminaphthorhodafluor-1. In HEPES-buffered medium, basal intracellular pH (pH(i)) was similar at 7.28+/-0.02 in neonatal and 7.31+/-0.02 in adult myocytes, but intrinsic buffering power was lower in the former age group. The rate at which pH(i) recovered from a similar acid load was significantly greater in neonatal than in adult myocytes (0.36+/-0.07 v 0.16+/-0.02 pH units/min at pH(i)=6.8). This was reflected by a significantly greater J(H) (22+/-4 v 9+/-1 pmol/cm2/s at pH(i)=6.8), indicating greater sarcolemmal NHE activity in neonatal myocytes. The concomitant reductions in tissue NHE-1 mRNA expression and sarcolemmal NHE activity suggest that myocardial NHE-1 is subject to regulation at the mRNA level during postnatal development.
Differential protein expression and subcellular distribution of TGFbeta1, beta2 and beta3 in cardiomyocytes during pressure overload-induced hypertrophy.
The transforming growth factor beta (TGFbeta) superfamily plays an important role in the myocardial response to hypertrophy. We have investigated the protein expression of TGFbeta1, beta2 and beta3 in left ventricular tissue, and determined their subcellular distribution in myocytes by immunoblotting and immunocytochemistry during the development of left ventricular hypertrophy (LVH), using isoform specific antibodies to TGFbeta1, beta2 and beta3. LVH was produced in rats by aortic constriction (AC) and LV tissue was obtained at days (d)0, 1, 3, 7, 14, 21 and 42 following operation. Compared with age matched sham-operated controls (SH), TGFbeta1 levels in LV tissue of AC rats increased significantly from d1-d14 (P<0.03) concomitant with the adaptive growth of LV tissue. In contrast, TGFbeta3 levels decreased in LV tissue of AC rats from d3 post-operation (significant from d14-d42, P<0.03). No significant difference in TGFbeta2 levels were observed from SH and AC rats after operation. Antibodies to TGFbeta1 stained intercalated disks, sarcolemmal membranes and cytoplasm, but not nuclei, of cardiomyocytes on LV sections from untreated and SH rats. However, a trans-localisation of TGFbeta1 to the nuclei of cardiomyocytes was observed in AC hearts. Antibodies to TGFbeta3 stained T tubules, cytoplasm and the nuclei of cardiomyocytes from untreated and SH rats. However, by d7 post-AC operation, TGFbeta3 expression was lost rapidly from nuclei of cardiomyocytes followed by a reduction in total TGFbeta3 immunofluorescence in myocytes. Antibodies to TGFbeta2 stained sarcolemmal membranes of cardiomyocytes from both SH and AC rats without significant difference between groups. Thus, the differential pattern of protein expression and subcellular distribution of TGFbeta1, beta2 and beta3 in myocytes during the development of LVH suggests that these molecules play different roles in the response of cardiomyocytes to LVH.
Phorbol ester, but not ischemic preconditioning, activates protein kinase D in the rat heart.
The signal transduction pathways that mediate the cardioprotective effects of ischemic preconditioning remain unclear. Here we have determined the role of a novel kinase, protein kinase D (PKD), in mediating preconditioning in the rat heart. Isolated rat hearts (n=6/group) were subjected to either: (i) 36 min aerobic perfusion (control); (ii) 20 min aerobic perfusion plus 3 min no-flow ischemia, 3 min reperfusion, 5 min no-flow ischemia, 5 min reperfusion (ischemic preconditioning); (iii) 20 min aerobic perfusion plus 200 nmol/l phorbol 12-myristate 13-acetate (PMA) given as a substitute for ischemic preconditioning. The left ventricle then was excised, homogenized and PKD immunoprecipitated from the homogenate. Activity of the purified kinase was determined following bincubation with [gamma32P]-ATP+/-syntide-2, a substrate for PKD. Significant PKD autophosphorylation and syntide-2 phosphorylation occurred in PMA-treated hearts, but not in control or preconditioned hearts. Additional studies confirmed that recovery of LVDP was greater and initiation of ischemic contracture and time-to-peak contracture were less, in ischemic preconditioned hearts compared with controls (P<0.05). Our results suggest that the early events that mediate ischemic preconditioning in the rat heart occur via a PKD-independent mechanism.
Expression and activities of cyclins and cyclin-dependent kinases in developing rat ventricular myocytes.
The molecular mechanisms responsible for the alterations in proliferative capacity of cardiac myocytes during development remain unknown; however, cell cycle dependent molecules may be involved. We have determined the expression of cyclins A, D1-3 and E, and cyclin-dependent kinases (CDKs) 2, 4, 5 and 6 and cdc2 in freshly isolated rat cardiac myocytes from fetal (18 days gestation), neonatal (2 days post-natal) and adult animals by immunoblotting. Our results show a dramatic decrease in expression of these proteins during normal cardiac development, such that levels are highest in fetal myocytes but are significantly down-regulated in adult cells (P<0.05, in each case). We also have determined the in vitro kinase activities of cdc2, CDK2, CDK4, CDK5 and CDK6 immunocomplexes in fetal, neonatal and adult myocytes. There was a consistent and significant loss of cdc2, CDK2, CDK4 and CDK6 kinase activities in adult cardiac cell lysates (5.3-, 10.6-, 1.5- and 1.9-fold decreases, respectively) when compared to neonatal samples (P<0.05); CDK5 activity showed a similar trend but failed to reach significance. In conclusion, our results show that the expression and activities of various positive regulators of the cell cycle are down-regulated significantly during development of the cardiac myocyte, concomitant with the loss of proliferative capacity in adult myocytes. Down-regulation of these proteins may be pivotal in the withdrawal of the cardiac myocyte from the cell cycle.
Expression and regulation of 80K/MARCKS, a major substrate of protein kinase C, in the developing rat heart.
OBJECTIVE: Protein kinase C (PKC) plays a pivotal role in modulating the growth and differentiation of many cell types including the cardiac myocyte. However, little is known about molecules that act immediately downstream of PKC in the heart. In this study we have investigated the expression of 80K/MARCKS, a major PKC substrate, in whole ventricles and in cardiac myocytes from developing rat hearts. METHODS: Poly A/ RNA was prepared from neonatal (2-day) and adult (42-day) cardiac myocytes and whole ventricular tissue and mRNA expression determined by reverse transcription-polymerase chain reaction (RT-PCR) using primers designed to identify a 420 bp fragment in the 80K/MARCKS gene. Protein extracts were prepared from either 2-day and 42 day cardiac myocytes or from whole ventricular tissue at 2, 5-11, 14, 17, 21, 28 and 42 days of age. Protein expression was determined by immunoblotting with an 80/MARCKS antipeptide antibody and PKC activity was determined by measuring the amount of gamma 32 P-ATP transferred to a specific peptide substrate. RESULTS: RT-PCR analysis of 80K/MARCKS mRNA in neonatal (2-day) and adult (42-day) cardiac myocytes showed the expression of this gene in both cell types. Immunoblotting revealed maximum 80K/MARCKS protein expression in whole ventricular tissue at 5 days (a 75% increase above values at 2 days), followed by a transient decrease in expression during the 6-8 day period (61% of the protein expressed at 2 days for 8-day tissue) with levels returning to 5 day levels by 11 days of age. 80K/MARCKS protein was present in cardiac myocytes at 2 days of age whereas it was not detectable in adult cells. In addition, PKC activity levels increased to 160% of levels present at 2 days in 8-day old ventricles with PKC activity levels returning to 5-day levels by 9 days of age. This was then; followed by a steady decline in both 80K/MARCKS protein expression and PKC activity through to adulthood. CONCLUSIONS: Expression of the PKC substrate, 80K/MARCKS, in cardiac myocytes changes significantly during development and the transient loss of immunoreactive protein during the 6-8 day development period may reflect 80K/MARCKS phosphorylation and subsequent down-regulation as a result of the concomitant up-regulation of PKC activity at this time.
Downregulation of cyclin-dependent kinase inhibitors p21 and p27 in pressure-overload hypertrophy.
We postulated that the cyclin-dependent kinase inhibitors p21 and p27 could regulate the alterations in growth potential of cardiomyocytes during left ventricular hypertrophy (LVH). LVH was induced in adult rat hearts by aortic constriction (AC) and was monitored at days 0, 1, 3, 7, 14, 21, and 42 postoperation. Relative to sham-operated controls (SH), left ventricle (LV) weight-to-body weight ratio in AC increased progressively with time without significant differences in body weight or right ventricle weight-to-body weight ratio. Atrial natriuretic factor mRNA increased significantly in AC to 287% at day 42 compared with SH (P < 0.05), whereas p21 and p27 mRNA expression in AC rats decreased significantly by 58% (P < 0.03) and 40% (P < 0.05) at day 7, respectively. p21 and p27 protein expression decreased significantly from days 3 to 21 in AC versus SH, concomitant with LV adaptive growth. Immunocytochemistry showed p21 and p27 expression in cardiomyocyte nuclei. Thus downregulation of p21 and p27 may modulate the adaptive growth of cardiomyocytes during pressure overload-induced LVH.
MARCKS functions as a novel growth suppressor in cells of melanocyte origin.
Protein kinase C (PKC) plays a pivotal role in modulating the growth of melanocytic cells in culture. We have shown previously that a major physiological substrate of PKC, the 80 kDa myristoylated alanine-rich C-Kinase substrate (MARCKS), can be phosphorylated in quiescent, non-tumorigenic melanocytes exposed transiently to a biologically active phorbol ester, but cannot be phosphorylated in phorbol ester-treated, syngeneic malignant melanoma cells. Despite its ubiquitous distribution, the function of MARCKS in cell growth and transformation remains to be demonstrated clearly. We report here that MARCKS mRNA and protein levels are down-regulated significantly in the spontaneously derived murine B16 melanoma cell line compound with syngeneic normal Mel-ab melanocytes. In contrast, the tumourigenic v-Ha-ras-transformed melanocytic line, LTR Ras 2, showed a high basal level of MARCKS phosphorylation which was not enhanced by treatment of cells with phorbol ester. Furthermore, protein levels of MARCKS in LTR Ras 2 cells were similar to those expressed in Mel-ab melanocytes. However, in four out of six murine tumour cell lines investigated, levels of MARCKS protein were barely detectable. Transfection of B16 cells with a plasmid containing the MARCKS cDNA in the sense orientation produced two neomycin-resistant clones displaying reduced proliferative capacity and decreased anchorage-independent growth compared with control cells. In contrast, transfection with the antisense MARCKS construct produced many colonies which displayed enhanced growth and transforming potential compared with control cells. Thus, MARCKS appears to act as a novel growth suppressor in the spontaneous transformation of cells of melanocyte origin and may play a more general role in the tumour progression of other carcinoma.
Role of protein kinase C in ischemic preconditioning: player or spectator?
Explore the source record for details and available documents.
Inhibitors of bacterial signal peptidase: a series of 6-(substituted oxyethyl)penems.
A series of 6-(substituted oxyethyl)penem esters having the (5S) stereochemistry which are potent inhibitors of Escherichia coli leader peptidase is described. Structure-activity relationships are discussed.
A comparison of magnesium sulfate and indomethacin to magnesium sulfate only for tocolysis in preterm labor with advanced cervical dilation.
Preterm labor becomes more difficult to inhibit as the degree of cervical dilation increases. Indeed, some physicians do not even attempt tocolysis with advanced cervical dilation. We compared single- versus double-agent tocolytic therapy when the cervix was dilated 3 cm or greater. We conducted a retrospective study of 44 patients with preterm labor of unknown etiology and with cervical dilation of greater than 3 cm. At the admitting physician's discretion, patients were treated with either magnesium sulfate or with magnesium sulfate and indomethacin in combination. Longer duration of successful tocolysis was noted in the group that received both magnesium sulfate and indomethacin (368.3 hours versus 70.9 hours). No maternal complications occurred in either group. These pilot data suggest that tocolysis with magnesium sulfate and indomethacin is a safe, effective method of tocolysis in patients with advanced cervical dilation.