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Biomedical subjects

S MacKenzie

Publications and source records attributed to S MacKenzie.

At least 19 recordsLinked to original sources
PubMed2005

Transcriptional analysis of LPS-stimulated activation of trout (Oncorhynchus mykiss) monocyte/macrophage cells in primary culture treated with cortisol.

Primary immune responses to pathogen invasion are mediated by the innate immune system in which tissue macrophages play a key role. During infectious processes glucocorticoids generally may function to dampen inflammatory responses. In this study, the ability of cortisol to directly modulate the transcriptional response of rainbow trout macrophages to the cellular activator lipopolysaccharide (LPS) was investigated. The results indicate that cortisol significantly inhibits the well-described LPS-dependent induction of the expression of TNF-alpha2, a pro-inflammatory cytokine. In order to further characterize the molecular effects of LPS and the immunomodulatory role of cortisol, the in vitro macrophage response to LPS in the absence or presence of 12-h cortisol exposure was analyzed utilizing a salmonid-specific microarray platform. Genes that were stimulated or inhibited with LPS plus cortisol fell into several major functional groups. The first, a general "response" group comprising genes within ontology classes including the response to external stimuli, stress, humoral immunity and apoptosis, exhibited a significant increase after LPS stimulation, whereas suppression of this response was observed in the presence of cortisol. LPS stimulated other genes in a second group involved in cell signalling and also genes in a third group involved in the activation of transcription. Categories activated with cortisol were mainly related to various aspects of metabolism (including protein biosynthesis, binding and transport of ions) and structural proteins (mainly cytoskeleton and microtubules). The immunomodulatory action of cortisol on LPS-stimulated macrophages therefore appears more complex than simply the antagonism of LPS-induced transcriptional responses.

Animals↗
PubMed2005

Control of adipose tissue lipid metabolism by tumor necrosis factor-alpha in rainbow trout (Oncorhynchus mykiss).

Tumor necrosis factor-alpha (TNF alpha) is a cytokine with multiple biological functions which, in mammals, has been shown to modulate muscle and adipose tissue metabolism. In fish, TNF alpha has been identified in several species. However, few studies have examined the role of TNF alpha in fish outside the immune system. In this study, we assessed the effects of human recombinant TNF alpha and conditioned media from rainbow trout lipopolysaccharide (LPS)-stimulated macrophages (LPS-MCM) on lipolysis in isolated rainbow trout adipocytes. Furthermore, we studied the effects of an LPS injection in vivo on lipid metabolism. In our study, human recombinant TNF alpha stimulated lipolysis in trout adipocytes in a time- and dose-dependent manner. Similarly, LPS-MCM stimulated lipolysis in trout adipocytes when compared with control conditioned medium. Experiments using specific inhibitors of the MAP kinase pathway showed that p44/42 and p38 are partially involved in the lipolytic effects of TNF alpha. On the other hand, adipocytes from LPS-injected rainbow trout showed higher basal lipolysis than adipocytes from control fish after 24 h, while this effect was not seen at 72 h. Furthermore, lipoprotein lipase (LPL) activity in adipose tissue of LPS-injected fish was lower than in the controls at 24 h. These data suggest that TNF alpha plays an important role in the control of lipid metabolism in rainbow trout by stimulating lipolysis in vitro and in vivo and by down-regulating LPL activity of adipose tissue in vivo.

Adipose Tissue↗
PubMed2004

Impact of video colonoscopy on stage and outcome of patients with symptomatic colorectal cancer.

BACKGROUND: Double-contrast barium enema still is regarded by many as the investigation of choice for patients with large bowel symptoms. The aim of this study was to compare the stage and outcome of patients with colorectal cancer diagnosed by video colonoscopy (VC) and barium enema (BE) in a single institution. METHODS: Between July 1997 and December 2001, data were gathered prospectively in a series of 489 patients presenting consecutively with symptomatic colorectal cancer. Selection of patients for either VC or BE investigation was made by the clinician who examined the patient in the clinic. Of the 489 patients, 82 were excluded because they presented acutely or other methods were used for the diagnosis. RESULTS: A diagnosis of colorectal cancer was determined by VC for 292 patients and by BE for 115 patients. The patients in both groups were similar in terms of age, gender, and site of disease. Stage 1 disease (T1/2NO) was diagnosed for 87 (29.8%) patients in the VC group, as compared with 10 (8.7%) in the BE group (p < 0.0001). Early colorectal cancer (T1) was diagnosed for 43 patients in the VC group as compared to 1 patient in the BE group (p < 0.0001). During a median follow-up period of 33 months, 8.2% of the patients in the VC group had experienced recurrence after curative resection, as compared with 17.4% of the patients in the BE group p = 0.018). Freedom from disease (p = 0.02) and overall survival (p = 0.03) were significantly increased in the VC group. CONCLUSIONS: Videocolonoscopy used as the investigation of choice for patients with large bowel symptoms detects colorectal cancer at an earlier stage and has a significant impact on the outcome for this condition.

Aged↗
PubMed2003

Randomized clinical trial comparing consultant-led or open access investigation for large bowel symptoms.

BACKGROUND: Referral of patients with large bowel symptoms is common and increasing. Currently most of these referrals are assessed at an outpatient clinic to determine the need and priority for investigation. METHODS: Over 21 months, 1131 patients referred by the general practitioner with large bowel symptoms were randomized. Patients in the consultant-led group were assessed by surgeons with a colorectal interest while those in the open access group underwent colonoscopy if they were 55 years or older and flexible sigmoidoscopy if younger. RESULTS: The most common symptom among referred patients was rectal bleeding (69.1 per cent) followed by change in bowel habit (48.8 per cent) and abdominal pain (32.3 per cent). There was a significant trend (P < 0.001) for patients in the consultant-led to have more investigations, and more patients in this group had no investigations (P < 0.001). Despite this, the percentage of patients with colonic or other pathology diagnosed was the same in both groups, 63.6 per cent in the consultant-led group compared with 61.8 per cent in the open access group (P = 0.558). Likewise the percentage of patients with cancer or other significant pathology was similar in both groups (13.9 versus 15.4 per cent; P = 0.532). The mean(s.d.) time to diagnose cancer or other significant pathology was 55.1(39.2) days in the consultant-led group compared with 57.4(33.6) days in the open access group (P = 0.514). The cost per patient was almost pound 105 more for patients in the consultant-led group. CONCLUSION: Patients referred by the general practitioner with large bowel symptoms should go directly to a properly managed and staffed open access large bowel investigation unit. This would enable most patients to have their investigations completed at one hospital attendance.

Ambulatory Care↗
PubMed2002

The effects of dietary sodium loading on the activity and expression of Na, K-ATPase in the rectal gland of the European dogfish (Scyliorhinus canicula).

cDNA fragments of both the alpha- and beta-subunits of the Na, K-ATPase and a cDNA fragment of the secretory form of Na-K-Cl cotransporter from the European dogfish (Scyliorhinus canicula) were amplified and cloned using degenerate primers in RT-PCR. These clones were used along with a sCFTR cDNA from the related dogfish shark, Squalus acanthias to characterise the expression of mRNAs for these ion transporters in the dogfish rectal gland subsequent to an acute feeding episode. Following a single feeding event where starved dogfish were fed squid portions (20 g squid/kg fish), there was a delayed and transient 40-fold increase in the activity of Na, K-ATPase in crude rectal gland homogenates. Increases in enzyme activity were apparent 3 h after the feeding event and peaked at 9 h before returning to control values within 24 h. These increases in activity were accompanied by small and transient decreases in plasma sodium and chloride concentrations lasting up to 3 days. Significant increases in the expression of mRNAs for alpha- and beta-subunits of the Na, K-ATPase, the Na-K-Cl cotransporter and CFTR chloride channel were detected but not until 1-2 days after the feeding event. It is concluded that the transient increase in Na, K-ATPase activity is not attributable to increases in the abundance of alpha- and beta-subunit mRNAs but must be associated with some, as yet unknown, post-transcriptional activation mechanism.

Amino Acid Sequence↗
PubMed2001

Sequencing and characterization of the human thiazide-sensitive Na-Cl cotransporter (SLC12A3) gene promoter.

The thiazide-sensitive Na-Cl cotransporter SLC12A3 displays expression restricted to distal convoluted tubule cells where it catalyzes the uptake of sodium and chloride through the apical membrane. We sequenced 1959 bp of the 5' flanking region of human SLC12A3, located the area of transcription initiation, and used deletion constructs of the flanking region to determine areas that affect reporter gene expression in two cell lines, MDCT and CHO. Amplification of the 5' end of SLC12A3 cDNA from an adapter-ligated human kidney cDNA library demonstrated that transcription initiation is confined to an area from -18 to -6 bp upstream of the translation start codon. Maximum promoter activity (9.815 +/- 0.864 times control) was observed in MDCT cells using a promoter containing 1019 bp of the 5' flanking region. A promoter containing only 134 bp of the 5' flanking region upstream of the translation initiation codon maintained reporter gene expression at levels equal to 75% of that maximally observed (7.375 +/- 0.533 times control). Sequence analysis of this minimal promoter responsible for most of the SLC12A3 promoter activity revealed a TATA element, two Sp binding sites, a potential E box, and a potential binding site for NF-1/CTF or NY-I/CP-I. This promoter, and all other promoter constructs from SLC12A3, displayed repressor activity in CHO cells. A construct containing sequence 94 bp upstream of the initiation codon with two potential Sp binding sites was required for this repression. Protein-DNA interactions between the 182 bp region immediately upstream of the start codon and the nuclear proteins from rat kidney cortex and HeLa cells were examined to further clarify the role of the putative binding sites for SLC12A3 expression. Physiological studies investigating the effects of osmolarity, pH, and mineralocorticoid steroid on promoter activity demonstrated that the promoter activity was repressed by acidification, whereas no effects of increased osmolarity or deoxycorticosterone acetate addition were observed.

5' Untranslated Regions↗
PubMed2001

Cardiac troponin I is modified in the myocardium of bypass patients.

BACKGROUND: Selective proteolysis of cardiac troponin I (cTnI) is a proposed mechanism of contractile dysfunction in stunned myocardium, and the presence of cTnI degradation products in serum may reflect the functional state of the remaining viable myocardium. However, recent swine and canine studies have not demonstrated stunning-dependent cTnI degradation. METHODS AND RESULTS: To address the universality of cTnI modification, myocardial biopsy samples were obtained from coronary artery bypass patients (n=37) before and 10 minutes after removal of cross-clamp. Analysis of biopsy samples for cTnI by Western blotting revealed a spectrum of modified cTnI products in myocardium both before and after cross-clamp, including degradation products (7 products resulting from differential N- and C-terminal processing) and covalent complexes (3 products). In particular, a 22-kDa cTnI degradation product with C-terminal proteolysis was identified, which may represent an initial ischemia-dependent cTnI modification, similar to cTnI(1-193) observed in stunned rat myocardium. Although no systematic change in amount of modified cTnI was observed, subgroups of patients displayed an increase (n=10, 85+/-5% of cTnI remaining intact before cross-clamp versus 75+/-5% after) or a decrease (n=12, 67+/-5% before versus 78+/-5% after). Electron microscopy demonstrated normal ultrastructure in biopsy samples, which suggests no necrosis was present. In addition, cTnI modification products were observed in serum through a modified SDS-PAGE methodology. CONCLUSIONS: cTnI modification, in particular proteolysis, occurs in myocardium of bypass patients and may play a key role in stunning in some bypass patients.

Biopsy↗
PubMed2001

Integrating signals from T-cell receptor and serum by T cells enhance translation of tumour necrosis factor-alpha.

Tumour necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine produced by several cell types, including T cells upon antigen stimulation. Its production is crucial for the development of an early defence against many pathogens, but its beneficial effects are dependent on the strength and duration of its expression. In this paper we present evidence indicating that serum increases translational efficiency of TNF-alpha in human peripheral blood mononuclear cells stimulated with superantigen. The increase in translation of TNF-alpha due to serum could be inhibited by the phosphatidylinositol (PI) 3-K inhibitors, wortmannin and LY294002, suggesting that PI 3-K is involved in the translational control of TNF-alpha by serum. Similarly to primary T cells, stimulation of Jurkat T cells with superantigen led to TNF-alpha secretion and this was up-regulated by serum. Transfection of Jurkat cells with a constitutively active form of PI 3-Kalpha increased the production of TNF-alpha in cells stimulated with superantigen. Additionally, we used the specific inhibitors targeting ERK kinase and p38 mitogen-activated protein kinase (MAPK), potentially downstream of PI 3-kinase, PD98059 and SB203580. Differently from with PI 3-K inhibitors, the accumulation of TNF-alpha mRNA was inhibited by PD98059 or SB203580. These results suggest that, in T cells, activation of PI 3-K is an important step in controlling TNF-alpha protein synthesis in response to growth factors.

CD4-Positive T-Lymphocytes↗
PubMed2001

High-throughput interpretation of pathways and biology.

The genomics revolution is opening many diverse pathways in science. In the midst of the tidal wave of information being generated by the new "omics" disciplines, bioinformatics is meeting the challenge to decipher and organize the data mountain. The endeavor to integrate diverse biological data into a common vocabulary will be an essential part of the "biotechnological age" leading to a new understanding of complex biological systems. This potential promises to revolutionize medicine as knowledge is translated into novel therapies. The Integrated Bioinformatics meeting held in Zurich January 2426, 2001, provided an insight into the cutting edge of bioinformatics and its central role in the genomics revolution. The evolution of bioinformatics will act as a guide in 21st century biological sciences.

Journal Article↗
PubMed2000

Corticosteroid regulation of amiloride-sensitive sodium-channel subunit mRNA expression in mouse kidney.

Corticosteroid control of distal nephron sodium handling, particularly through the amiloride-sensitive sodium channel (ENaC), has a key role in blood pressure regulation. The mechanisms regulating ENaC activity remain unclear. Despite the generation of useful mouse models of disorders of electrolyte balance and blood pressure, there has been little study of distal nephron sodium handling in this species. To investigate how corticosteroids regulate ENaC activity we isolated cDNA for the three mouse ENaC subunits (alpha, beta and gamma), enabling their quantitation by competitive PCR and in situ hybridisation. Kidneys were analysed from mice 6 days after adrenalectomy or placement of osmotic mini-pumps delivering aldosterone (50 microg/kg per day), dexamethasone (100 microg/kg per day), spironolactone (20 mg/kg per day) or vehicle alone (controls). In controls, renal ENaCalpha mRNA exceeded beta or gamma by approximately 1.75- to 2.8-fold. All subunit mRNAs were expressed in renal cortex and outer medulla, where the pattern of expression was fully consistent with localisation in collecting duct, whereas the distribution in cortex suggested expression extended beyond the collecting duct into adjacent distal tubule. Subunit mRNA expression decreased from cortex to outer medulla, with a gradual reduction in beta and gamma, and ENaCalpha decreased sharply ( approximately 50%) across the outer medulla. Expression of ENaCbeta and gamma (but not alpha) extended into inner medulla, suggesting the potential for inner medulla collecting duct cation channels in which at least ENaCbetagamma participate. Aldosterone significantly increased ENaC subunit expression; the other treatments had little effect. Aldosterone caused a 1.9- to 3.5-fold increase in ENaCalpha (particularly marked in outer medullary collecting duct), but changes for beta and gamma were minor and limited to the cortex. The results raise the possibility that medullary ENaCalpha upregulation by aldosterone will create more favourable subunit stoichiometry leading to a more substantial increase in ENaC activity. In cortex, such a mechanism is unlikely to have a major role.

Adrenal Cortex Hormones↗
PubMed1999

Construction and characterization of a common bean bacterial artificial chromosome library.

We have constructed a common bean (Phaseolus vulgaris L.) bacterial artificial chromosome (BAC) library consisting of 33,792 clones and an estimated 3- to 5-fold coverage of the common bean genome. Leaf nuclei were used as the source for high-molecular-weight DNA, and an endonuclease/methylase competition assay was employed to partially cleave the DNA. The library was screened with a number of nuclear and mitochondrial probes. Each nuclear probe identified at least two BACs with an average insert size of ca. 100 kb. Only 26 clones were identified after hybridizing with mitochondrial probes, indicating contamination with organellar sequences is low. Numerous clones could be identified after screening the library with two repetitive probes flanking the nuclear fertility restorer Fr. Intriguingly, 12 clones appeared to hybridize to both markers, and restriction analysis of these clones revealed that they can be assembled into maximally four contigs, suggesting that these repetitive probes may be useful for the physical mapping of the Fr locus.

Chromosomes, Bacterial↗
PubMed1998

One hundred seventy-fold increase in excretion of an FV fragment-tumor necrosis factor alpha fusion protein (sFV/TNF-alpha) from Escherichia coli caused by the synergistic effects of glycine and triton X-100.

To target tumor necrosis factor alpha (TNF-alpha) to tumor cells, recombinant DNA techniques were used to construct and express the fused gene VKLVH-TNF-alpha, which encodes the secreted form of single-chain fusion protein sFV/TNF-alpha in Escherichia coli. sFV/TNF-alpha was secreted into the culture medium and purified by affinity chromatography. The production of the fusion protein in the culture medium under the optimal conditions of 30 degrees C and 37 micromol of isopropyl-beta-D-thiogalactopyranoside (IPTG) per liter was 16- and 5-fold higher than that under the standard conditions of 37 degrees C and 1 mmol of IPTG per liter. Fusion protein excretion into culture medium with 2% glycine, 1% Triton X-100, or both of these two chemicals was either 14-, 38-, or 170-fold higher, respectively than that without the two chemicals. The final yield of sFV/TNF-alpha was estimated to be 50 mg/liter. The loss of integrity of the cellular membrane may be a potential mechanism for enhancement of fusion protein production and excretion by treatment with glycine and Triton X-100. This study thus provides a practical, large-scale method for more efficient production of the heterologous fusion protein sFV/TNF-alpha in E. coli by using glycine and Triton X-100.

Culture Media↗
PubMed1998

Mechanism of epinephrine-induced platelet aggregation.

We report that a genetic polymorphism of the alpha2-adrenergic receptor (A2AR) encoded by chromosome 10 is associated with hypertension and an increase in epinephrine-mediated platelet aggregation in humans. The mechanism responsible for this heritable contrast in sensitivity to epinephrine is unknown. We tested our hypothesis that epinephrine-induced platelet aggregation is mediated by activation of chloride transport. We measured epinephrine-mediated increases in optical density of gel-filtered platelets suspended in a bicarbonate-buffered physiological salt solution. Compared with platelets incubated in the control buffer (130 mmol/L NaCl), platelets incubated with either bumetanide, a Na/K/2Cl cotransport inhibitor; anthracene-9-carboxylic acid, a chloride channel blocker; or acetazolamide, an agent that blocks ATP-dependent chloride transport had significantly decreased aggregation responses to epinephrine. When measured fluorometrically, epinephrine significantly increased intraplatelet chloride concentrations. Chloride-dependent modifications of epinephrine-induced platelet aggregation were not attributable to changes in A2AR ligand binding characteristics or to the concentration of platelet cAMP. Finally, subthreshold concentrations of epinephrine also potentiated thrombin-induced platelet aggregation, and blockade of chloride transport diminished this synergistic action of epinephrine on thrombin-stimulated platelet aggregation. Heritable differences in epinephrine-mediated platelet aggregation may be attributable to genetic differences in chloride transport in platelets. Furthermore, because we observed a necessary role for chloride transport in epinephrine-mediated platelet aggregation, pharmacological agents that block chloride transport, such as diuretics, may provide salutary protection against vascular thrombosis in patients with hypertension independent of the effect of these drugs on blood pressure.

Adrenergic alpha-Antagonists↗
PubMed1997

Growth hormone and phorbol esters require specific protein kinase C isoforms to activate mitogen-activated protein kinases in 3T3-F442A cells.

Previous studies have shown that the activation of p44 and p42 mitogen-activated protein (MAP) kinases (ERK1 and ERK2) by growth hormone (GH) and phorbol esters, but not by epidermal growth factor, in 3T3-F442A preadipocytes is dependent on protein kinase C (PKC). In the present study two approaches have been taken to determine the PKC isoform dependence of MAP kinase activation in these cells. By immunoblotting with specific antibodies, the cells were found to express PKC-alpha, -gamma,-delta, -epsilon and -zeta. Treatment of cells with 500 nM PMA for 3 h led to the complete depletion of PKC-delta and the partial depletion of PKC-alpha but did not significantly affect the expression of the other PKC isoforms. In parallel, such treatment severely attenuated the ability of GH to activate MAP kinase. The degree of this attenuation was not increased by more prolonged PMA pretreatment, indicating that PKC-delta and perhaps PKC-alpha are important for MAP kinase activation by GH. These experiments further revealed that additional PKC isoforms were required for the full activation of MAP kinases by acute treatment with PMA. A second approach involved the use of anti-sense oligodeoxynucleotides (ODNs) to deplete the individual PKC isoforms selectively. Each of the ODNs used effectively depleted the relevant isoform to undetectable levels and did not affect the expression of the other PKC isoforms. Pretreatment of cells with PKC-delta anti-sense ODN, but not with anti-sense ODN to the other phorbol ester-sensitive isoforms, severely attenuated the activation of MAP kinases by GH. PKC-delta anti-sense ODN also blocked (by approx. 50%) the activation of MAP kinases by PMA. Furthermore a combination of PKC-delta and -epsilon anti-sense ODNs completely blocked the effect of PMA on MAP kinases. Collectively, these results indicate that the novel PKC-delta and -epsilon isoforms can couple to the MAP kinase pathway in 3T3-F442A cells but that the activation of MAP kinases by GH specifically involves PKC-delta.

3T3 Cells↗
PubMed1996

Endotoxin-induced adhesion of red blood cells to pulmonary artery endothelial cells.

Cell-cell interactions are important in intravascular inflammation. Neutrophils and monocytes adhere to the vascular endothelium and release mediators, such as tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta, and reactive oxygen species. Red blood cells (RBC) from patients with malaria, sickle cell anemia, and diabetes also adhere to endothelial cells. The objectives of this investigation were to develop a bovine system of RBC adhesion to endothelial cells and to begin to investigate the mechanisms involved in the RBC adhesion. We show that 51Cr-RBC adhere to bovine pulmonary artery endothelial cells (BPAEC) after stimulation of both cell types with endotoxin (ETX; 50 micrograms/ml). RBC adhesion to BPAEC depended on the ETX concentration and the presence of divalent cations. TNF-alpha, IL-1 beta, and antioxidants (superoxide dismutase; catalase; and dimethyl sulfoxide) all induced RBC adhesion to BPAEC. Phosphatidylserine, which has been implicated in adhesion of sickle cells and aged RBC to endothelium, reduced RBC adhesion to BPAEC, whether ETX-treated or not. In conclusion, ETX, proinflammatory cytokines and, surprisingly, antioxidants increase RBC adherence to BPAEC monolayers. RBC adhesion to endothelium is decreased by phosphatidylserine.

Animals↗