Biomedical subjects

S Daniel

Publications and source records attributed to S Daniel.

At least 37 records · Page 2Linked to original sources

PubMed2001

Leukocyte trafficking and myocardial reperfusion injury in ICAM-1/P-selectin-knockout mice.

P-selectin and intercellular adhesion molecule-1 (ICAM-1) mediate early interaction and adhesion of neutrophils to coronary endothelial cells and myocytes after myocardial ischemia and reperfusion. In the present study, we examined the physiological consequences of genetic deletions of ICAM-1 and P-selectin in mice. In wild-type mice, after 1 h of ischemia followed by reperfusion, neutrophil influx into the area of ischemia was increased by 3 h with a peak at 24 h and a decline by 72 h. ICAM-1/P-selectin-deficient mice showed a significant reduction in neutrophils by immunohistochemistry or by myeloperoxidase activity at 24 h but no significant difference at 3 h. Infarct size (area of necrosis/area at risk) assessed 24 h after reperfusion was not different between wild-type and deficient mice after 30 min and 1 h of occlusion. Mice with a deficiency in both ICAM-1 and P-selectin have impaired neutrophil trafficking without a difference in infarct size due to myocardial ischemia-reperfusion.

Animals↗

PubMed2001

Optimized T7 amplification system for microarray analysis.

Glass cDNA microarray technologies offer a highly parallel approach for profiling expressed gene sequences in disease-relevant tissues. However, standard hybridization and detection protocols are insufficient for milligram quantities of tissue, such as those derived from needle biopsies. Amplification systems utilizing T7 RNA polymerase can provide multiple cRNA copies from mRNA transcripts, permitting microarray studies with reduced sample inputs. Here, we describe an optimized T7-based amplification system for microarray analysis that yields between 200- and 700-fold amplification. This system was evaluated with both mRNA and total RNA samples and provided microarray sensitivity and precision that are comparable to our standard production process without amplification. The size distributions of amplified cRNA ranged from 200 bp to 4 kb and were similar to original mRNA profiles. These amplified cRNA samples were fluorescently labeled by reverse transcription and hybridized to microarrays comprising approximately 10,000 cDNA targets using a dual-channel format. Replicate hybridization experiments were conducted with the same and different tissues in each channel to assess the sensitivity and precision of differential expression ratios. Statistical analysis of differential expression ratios showed the lower limit of detection to be about 2-fold within and between amplified data sets, and about 3-fold when comparing amplified data to unamplified data (99.5% confidence).

Biotechnology↗

PubMed2001

Parkinson's disease is not associated with the combined alpha-synuclein/apolipoprotein E susceptibility genotype.

A recent study showed significant association of sporadic Parkinson's disease with a polymorphism within the alpha-synuclein gene and closely linked DNA markers on chromosome 4q and the APOE epsilon4 allele. A combined alpha-synuclein/APOE-epsilon4 genotype increased the relative risk of developing Parkinson's disease 12-fold. We failed to confirm this association in a much larger sample of histopathologically proven cases of Parkinson's disease and controls.

Alleles↗

PubMed2001

Stenting of partial and total coronary occlusions in Trinidad and Tobago.

In order to evaluate the efficacy and safety of coronary stenting, we reviewed the first 32 consecutive patients (34 vessels) who underwent elective coronary stenting during the period August 1999 to August 2000 inclusive at the Digital Lab installed at the Eric Williams Medical Sciences Complex, Trinidad and Tobago. Aspirin, heparin and ticlopidine were used routinely. Abciximab was used in selected cases (38%). The mean age of patients was 55 +/- 10 years. Eighty-one per cent were male, 52% were hypertensive and 21% were diabetic. Sixty-five per cent had severe angina. Prior Coronary Artery Bypass Grafting (CABG) was performed in 3% and previous Percutaneous Transluminal Coronary Angioplasty (PTCA) in 3%. Multivessel disease was present in 43%. The mean left ventricular ejection fraction was 53 +/- 12%. The culprit lesion was located in either the native left anterior descending (LAD) coronary artery (53%), right coronary artery (RCA) (31%), circumflex artery 13% and saphenous vein graft (3%). The mean baseline diameter stenosis was 91 +/- 9% and this was reduced to 13 +/- 33% after stenting. Procedural success was 100% for 26 partially occluded vs 50% for 8 totally occluded vessels. For the total occlusions, procedural success was inversely related to the duration of the occlusion. There were no cases of death, acute vessel closure, Q-wave myocardial infarction, repeat PTCA or emergent Coronary Artery Bypass Graft (CABG) during and following the procedure. Distal embolization occurred in one patient. The mean duration of hospital stay was one day (for 30 outpatient cases). One patient had recurrence of symptoms with a negative stress test. No patient underwent repeat angiography during the first year of follow-up. Coronary stents were successfully implanted at a tertiary care facility in the Caribbean with low in-hospital morbidity and mortality. Stents markedly reduced the diameter stenosis of the coronary lesion during PTCA. The incidence of clinical restenosis was low. Coronary revascularisation can be successfully achieved by coronary stenting in the Caribbean.

Angioplasty, Balloon, Coronary↗

PubMed2001

High-throughput genomic and proteomic analysis using microarray technology.

BACKGROUND: High-density microarrays are ideally suited for analyzing thousands of genes against a small number of samples. The next step in the discovery process is to take the resulting genes of interest and rapidly screen them against thousands of patient samples, tissues, or cell lines to further investigate their involvement in disease risk or the response to medication. METHODS: We used a microarray technology platform for both single-nucleotide polymorphisms (SNPs) and protein expression. Each microarray contains up to 250 elements that can be customized for each application. Slides contained either a 16- or 96-microarray format (4000-24,000 elements per slide), allowing the corresponding number of samples to be rapidly processed in parallel. RESULTS: Results for SNP genotyping and protein profiling agreed with results of restriction fragment length polymorphism (RFLP) analysis or ELISA, respectively. Genotyping analyses, using the microarray technology, on large sample sets over multiple polymorphisms in the NAT2 gene were in full agreement with traditional methodologies, such as sequencing and RFLP analysis. The multiplexed protein microarray had correlation coefficients of 0.82-0.99 (depending on analyte) compared with ELISAs. CONCLUSIONS: The integrated microarray technology platform is adaptable and versatile, while offering the high-throughput capabilities needed for drug development and discovery applications.

Arylamine N-Acetyltransferase↗

PubMed2000

Multiple system atrophy/progressive supranuclear palsy: alpha-Synuclein, synphilin, tau, and APOE.

Article abstract-Alpha synuclein, tau, synphilin, and APOE genotypes were analyzed in patients with multiple system atrophy (MSA) and progressive supranuclear palsy (PSP) and controls. The predisposing effect of the tau insertion polymorphism to the development of PSP is confirmed. However, no effect of alpha-synuclein, synphilin, or APOE variability on the development of PSP, or of tau, alpha-synuclein, APOE, or synphilin gene variability on the development of MSA, are demonstrated.

Apolipoproteins E↗

PubMed2000

Hexamminecobalt(III) chloride inhibits glucose-induced insulin secretion at the exocytotic process.

Hexamminecobalt(III) (HAC) chloride was found to have a potent inhibitory effect on glucose-induced insulin secretion from pancreatic islets. HAC at 2 mm inhibited the secretion in response to 22.2 mm glucose by 90% in mouse islets. Perifusion experiments revealed that the first phase of insulin secretion was severely suppressed and that the second phase of secretion was completely abrogated. Removal of HAC from the perifusate immediately restored insulin secretion with a transient overshooting above the normal level. However, HAC failed to affect glucose-induced changes in d-[6-(14)C]glucose oxidation, levels of reduced forms of NAD and NADP, mitochondrial membrane potential, ATP content, cytosolic calcium concentration, or calcium influx into mitochondria. Furthermore, HAC inhibited 50 mm potassium-stimulated insulin secretion by 77% and 10 microm mastoparan-stimulated insulin secretion in the absence of extracellular Ca(2+) by 80%. The results of a co-immunoprecipitation study of lysates from insulin-secreting betaHC9 cells using anti-syntaxin and anti-vesicle-associated membrane protein antibodies for immunoprecipitation or Western blotting suggested that HAC inhibited disruption of the SNARE complex, which is normally observed upon glucose challenge. These results suggest that the inhibitory effect of HAC on glucose-induced insulin secretion is exerted at a site(s) distal to the elevation of cytosolic [Ca(2+)], possibly in the exocytotic machinery per se; and thus, HAC may serve as a useful tool for dissecting the molecular mechanism of insulin exocytotic processes.

Adenosine Triphosphate↗

PubMed2000

Serotonin transporter function is modulated by brain-derived neurotrophic factor (BDNF) but not nerve growth factor (NGF).

The serotonin transporter (5-HTT) regulates serotonergic neurotransmission by determining the magnitude and duration of serotonergic responses. We have recently described a polymorphism in the 5-HTT gene promoter (5-HTTLPR) which influences the function of the 5-HTT and is associated with several psychiatric disorders. Immortalized B lymphocytes express the 5-HTT, and a B lymphocyte line has been shown to express the receptor for brain-derived neurotrophic factor, trkB. Since brain-derived neurotrophic factor (BDNF) is a specific growth and differentiation factor for serotonergic neurons, we assessed whether BDNF is able to modulate 5-HTT function in B lymphoblasts. Nerve growth factor (NGF), another neurotrophin which acts via the trkA receptor, was also studied. Eight immortalized B lymphoblast lines were generated and genotyped for the 5-HTTLPR. After treatment with BDNF or NGF, 5-HT uptake and proliferation of the cell lines were assessed. Two of the B cell lines showed a dose-dependent reduction of 5-HT uptake after exposure to BDNF. Both of these cell lines were homozygous for the long allele of the 5-HTTLPR. NGF did not influence 5-HT uptake or cellular proliferation in any of the cell lines. Thus, BDNF but not NGF may influence 5-HT uptake in some B lymphocytes. The fact that regulation of the 5-HTT was observed preferentially in cells of the long/long genotype indicates that presence of a short allele confers reduced regulatory capacity on the 5-HTT. In conclusion, B lymphoblasts represent a practical model for functional regulation of the 5-HTT by neurotrophins in serotonergic neurons.

Alleles↗

PubMed2000

Disruption of heart sarcoglycan complex and severe cardiomyopathy caused by beta sarcoglycan mutations.

Two young males with limb-girdle muscular dystrophy (LGMD) resulting from sarcoglycan deficiency died at 27 (patient 1) and 18 years (patient 2) of severe cardiomyopathy. Genetic analysis showed that they were compound heterozygotes for mutations in the beta sarcoglycan gene. One of these mutations, an 8 bp duplication in exon 3, was common to both patients. The second mutation in patient 2 was a 4 bp deletion at the splice donor site of intron 2, not reported previously. Patient 2 had more severe heart and skeletal muscle defects with faster deterioration; no sarcoglycans were detected in his skeletal muscle. The second mutation in patient 1, inferred because the unaffected father carries the 8 bp duplication, was not found. In patient 1, both heart and skeletal muscle were analysed and showed reduction of all sarcoglycans in both tissues and incorrect localisation of alpha and gamma sarcoglycans in heart. Therefore mutations in one sarcoglycan gene can disrupt the entire sarcoglycan complex in both skeletal and cardiac muscle. Differing expression patterns of sarcoglycan components in heart and skeletal muscle could be the result of alternatively spliced transcripts in these tissues. By sequencing an alternative transcript, highly expressed in the heart and skeletal muscle of patient 1, we found an 87 bp cryptic exon not previously reported. Although cardiomyopathy can result from mutations in alpha and gamma sarcoglycans, we show for the first time that the condition can also be caused by mutations in the beta sarcoglycan gene. This report therefore expands the phenotype of sarcoglycanopathies and suggests that cardiac function in LGMD patients with defective sarcoglycan expression should be monitored.

Adolescent↗

PubMed2000

Significant increase of aortic root volume and commissural area occurs prior to aortic valve opening.

BACKGROUND AND AIM OF THE STUDY: The increased use of autologous, homologous or heterologous aortic root demands a detailed knowledge of its anatomy and function. The advent of 3-D digital sonomicrometry offered the opportunity to acquire precise information on the root and leaflet movements during the cardiac cycle. METHODS: Under cardiopulmonary bypass, sonomicrometry crystals were implanted in the aortic root and valve of eight sheep. Crystals were sutured at each commissure (n = 3), the top of the sinotubular junction (n = 3), lowest point of the annulus (n = 3), and leaflet tip (n = 3). 3-D coordinates of each crystal were recorded, together with left ventricular and aortic root pressures and ECG. When the animal had returned to a stable hemodynamic condition, the maximum and minimum distances between two crystals, and areas between three crystals, were calculated. Changes in root volume and leaflet position were time-related to the pressure changes. RESULTS: The most significant change between maximum and minimum distance between crystals during the cardiac cycle occurred at the commissural level. Similarly, the triangle defined by the three commissural crystals showed the greatest change in area (47%). The root volume increased by an average of 22%; about 40% of this increase occurred during the isovolumic phase. The aortic leaflets began to open before ejection. CONCLUSION: We postulate that aortic valve opening is initiated by the outward pull of the commissures. These findings should impact on aortic root surgery.

Animals↗

PubMed2000

The feasibility of a porcine model of acute coronary occlusion and reperfusion using off-pump coronary artery bypass grafting.

We developed an open-chest porcine model of acute coronary occlusion and surgical reperfusion, and attempted to prevent intra-operative ischaemic ventricular fibrillation (VF) by a Retrograde Intracoronary Glyceryl trinitrate (RIG) infusion into the occluded vessel. Five Yorkshire pigs (weight 50 +/- 1.1 kg), randomized into 3 groups, underwent median sternotomy under general anaesthesia. One pig (Group 1, control) underwent sternotomy and pericardiotomy only. Four pigs underwent acute left anterior descending (LAD) coronary occlusion. Two pigs were not reperfused (Group 2). Two pigs underwent surgical reperfusion (Group 3) via left internal mammary artery (LIMA) grafting to the LAD using the Off-Pump Coronary Artery Bypass (OPCAB) technique. Ischaemic injury was assessed using 7-lead electrocardiography (ECG) and transthoracic/epimyocardial echocardiography (ECHO). Group 1: transient intraoperative hypotension and VF occurred. Successful resuscitation and 10-week survival (until sacrifice) with normal left ventricular (LV) function was achieved. Group 2: there were ECG and ECHO evidence of acute LV ischaemic dysfunction in both pigs. The surviving pig had persistent anterior hypokinesis at 8 1/2 months. The other died intra-operatively following progressive ischaemic LV dysfunction despite resuscitative attempts. Group 3: the surviving pig had normal LV function at 8 months. Initial anterior LV akinesis normalized within 7 days. The other developed post-occlusion haemodynamic instability and died intra-operatively despite reperfusion. In this porcine model, acute LAD artery occlusion modified by the novel RIG infusion technique, followed by surgical reperfusion (OPCAB) is feasible. This model would facilitate further development of OPCAB surgical expertise and understanding of the pathophysiology of ischaemia-reperfusion injury.

Animals↗

PubMed1999

Tapasin enhances assembly of transporters associated with antigen processing-dependent and -independent peptides with HLA-A2 and HLA-B27 expressed in insect cells.

Assembly of HLA class I-peptide complexes is assisted by multiple proteins that associate with HLA molecules in loading complexes. These include the housekeeping chaperones calnexin and calreticulin and two essential proteins, the transporters associated with antigen processing (TAP) for peptide supply, and the protein tapasin which is thought to act as a specialized chaperone. We dissected functional effects of processing cofactors by co-expressing in insect cells various combinations of the human proteins HLA-A2, HLA-B27, beta(2)-microglobulin, TAP, calnexin, calreticulin, and tapasin. Stability at 37 degrees C and surface expression of class I dimers correlated closely in baculovirus-infected Sf9 cells, suggesting that these cells retain empty dimers in the endoplasmic reticulum. Both HLA molecules form substantial quantities of stable complexes with insect cell-produced peptide pools. These pools are TAP-selected cytosolic peptides for HLA-B27 but endoplasmic reticulum-derived, i.e. TAP-independent peptides for HLA-A2. This discrepancy may be due to peptide selection by human TAP which is much better adapted to the HLA-B27 than to the HLA-A2 ligand preferences. HLA class I assembly with peptides from TAP-dependent and -independent pools was enhanced strongly by tapasin. Thus, tapasin acts as a chaperone and/or peptide editor that facilitates assembly of peptides with HLA class I molecules independently of mediating their interaction with TAP and/or retention in the endoplasmic reticulum.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Backbone cyclic peptide antagonists, derived from the insect pheromone biosynthesis activating neuropeptide, inhibit sex pheromone biosynthesis in moths.

We describe an application of the backbone cyclization and cycloscan concept for the design and synthesis of pheromone biosynthesis activating neuropeptide (PBAN) antagonists capable of inhibiting sex pheromone biosynthesis in Heliothis peltigera female moths. Two backbone cyclic (BBC) sub-libraries were designed and synthesized. The structure of the first sub-library ([Arg27]PBAN27-33NH2, termed the Ser sub-library) was based on the active C-terminal hexapeptide sequence (Tyr-Phe-Ser-Pro-Arg-Leu-NH2) of PBAN1-33NH2, which was found to comprise its active core. The second sub-library ([Arg27, D-Phe30]PBAN27-33NH2, termed the D-Phe sub-library) was based on the sequence of the lead antagonist Arg-Tyr-Phe-(D)Phe-Pro-Arg-Leu-NH2. In both sub-libraries the Pro residue was replaced by an Nalpha(omega-amino-alkyl)Gly building unit having various lengths of the alkyl chain. All the cyclic peptides in each sub-library had the same primary sequence and the same location of the ring. The members of each library differed from each other by the bridge size and bridge chemistry. Screening of the two libraries for pheromonotropic antagonists resulted in the disclosure of four compounds that fully inhibited sex pheromone biosynthesis at 1 nmol and were devoid of agonistic activity. All antagonistic peptides originated from the D-Phe sub-library. Substitution of the D-Phe30 amino acid with a Ser resulted in a loss of antagonistic activity. Agonistic activities were exhibited by peptides from both sub-libraries.

Animals↗

PubMed1999

Subunit affinities and stoichiometries of the human papillomavirus type 11 E1:E2:DNA complex.

The association between the papillomavirus E1 and E2 proteins is an important regulatory interaction, imparting coordinated control of viral transcription and replication. Using fluorescence polarization, we have characterized the interactions between HPV-11 E1, HPV-11 E2, and DNA in solution at equilibrium. For these studies, two double-stranded fluorescein-labeled oligonucleotides were prepared. The first fluorescent oligonucleotide, designated Fl-E2BS and containing a single E2 binding-site palindrome (ACCGN6CGGT), was used to determine the affinity of E2 for its DNA binding site. HPV-11 E2 bound Fl-E2BS with an apparent Kd of 0.84 nM. Binding was saturable and consistent with a single class of noninteracting sites. The second oligonucleotide, designated Fl-E1E2BS, contained both E1 and E2 sites in sequence derived directly from the HPV-11 origin of replication. Under titration conditions identical to those used for Fl-E2BS, the E2 protein exhibited reduced affinity for Fl-E1E2BS (Kd > 100 nM). E1 binding to Fl-E1E2BS was of very low affinity. Addition of excess HPV-11 E1 to Fl-E1E2BS lowered the dissociation constant for the E2:Fl-E1E2BS interaction to 2 nM. This effect was not dependent upon ATP or magnesium ion. Fluorescence polarization and other data suggest formation of a complex containing six E1 molecules and a single dimer of E2 bound to a single Fl-E1E2BS oligonucleotide; E2 dissociation from the final complex did not occur. In summary, physical interaction between E1 and E2 increases the DNA binding affinity of each. The role of this energy coupling may be to promote origin-specific binding of both E1 and E2 to DNA.

Adenosine Triphosphate↗

PubMed1999

The healthy patient: empowering women in their encounters with the health care system.

Many women's expectancies when they assume the role of patient include the experiences of regression, helplessness, passivity and fear. This paper describes techniques for interrupting this negative set and for facilitating the development of a self-efficacious state in which the woman experiences herself as an active and informed participant in her encounters with medical personnel.

Adult↗

PubMed1999

Clinical genetics of familial progressive supranuclear palsy.

Recent studies have shown that progressive supranuclear palsy (PSP) could be inherited, but the pattern of inheritance and the spectrum of the clinical findings in relatives are unknown. We here report 12 pedigrees, confirmed by pathology in four probands, with familial PSP. Pathological diagnosis was confirmed according to recently reported internationally agreed criteria. The spectrum of the clinical phenotypes in these families was variable including 34 typical cases of PSP (12 probands plus 22 secondary cases), three patients with postural tremor, three with dementia, one with parkinsonism, two with tremor, dystonia, gaze palsy and tics, and one with gait disturbance. The presence of affected members in at least two generations in eight of the families and the absence of consanguinity suggests autosomal dominant transmission with incomplete penetrance. We conclude that hereditary PSP is more frequent than previously thought and that the scarcity of familial cases may be related to a lack of recognition of the variable phenotypic expression of the disease.

Adult↗

PubMed1999

Prospective evaluation of unexplained chronic liver transaminase abnormalities in asymptomatic and symptomatic patients.

OBJECTIVES: It is currently recommend to perform a liver biopsy for patients with chronically elevated liver function tests (LFT) of unknown etiology (marker negative). The necessity and benefits of these recommendations are unknown. The aims of this study were to determine the prevalence of marker-negative LFT in patients referred for evaluation of chronically elevated LFT; to determine the prevalence of diseases that may be associated with marker-negative abnormal LFT; and to assess whether a liver biopsy alters the management of such patients. METHODS: We conducted a prospective observational study of 1124 adults referred for evaluation of chronically elevated LFT. Patients who consented to a liver biopsy were eligible. Marker-negative abnormal LFT was defined as the absence of accepted serum markers for infectious, metabolic, autoimmune, or hereditary liver disease, the absence of a history of alcohol or hepatotoxic drug use, and the absence of signs of chronic liver disease. RESULTS: Eighty-one of 1124 eligible patients were marker-negative. Liver biopsies in the 81 marker-negative patients revealed: normal histology (eight), steatosis (41), steatohepatitis (26), fibrosis (four), and cirrhosis (two). All 73 abnormal liver biopsies had some degree of steatosis. There were no significant associations between histological findings and the presence of obesity (p = 0.13), hyperlipidemia (p = 0.4), or diabetes (p = 0.9). There were no significant associations when classifying patients by gender or by symptoms. CONCLUSION: In the setting of marker-negative elevated LFT, the most likely histological diagnosis is fatty metamorphosis of the liver with occasional associated fibrosis.

Adult↗

PubMed1999

Identification of the docked granule pool responsible for the first phase of glucose-stimulated insulin secretion.

The mechanisms underlying the first phase of glucose-stimulated insulin release, the deterioration of which marks the early stages of both type 1 and type 2 diabetes, are essentially unknown. Among many hypotheses, one holds that the first phase is due to a readily releasable pool of insulin-containing granules. We used current knowledge of the mechanisms of exocytosis and the proteins involved in docking granules at the plasma membrane to test this hypothesis. A docked pool of readily releasable granules was identified by immunoprecipitation of the plasma membrane protein syntaxin with a specific antibody and by co-immunoprecipitation of soluble N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25) and the granule proteins synaptobrevin and synaptotagmin. The four SNARE proteins co-immunoprecipitated each other, thus identifying the core complex associated with docked granules. Using co-immunoprecipitation as a marker for docked granules, we found that the docked pool was rapidly discharged during the first phase of glucose-stimulated insulin release and refilled during the second phase. Other secretagogues also released the pool, whereas the physiological inhibitor norepinephrine blocked its release. Further studies on the nature of this pool of granules should shed light on the causes of its deterioration in the early stages of diabetes and the reasons for deficient insulin release.

Animals↗