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Biomedical subjects

A Jacob

Publications and source records attributed to A Jacob.

At least 55 records · Page 3Linked to original sources
PubMed1997

Dyspnea after pneumonectomy: the result of an atrial septal defect.

We report the case of severe hypoxemia attributable to right-to-left shunting through an atrial septal defect after right-sided pneumonectomy that developed in a 70-year-old man. Normal right atrial and pulmonary artery pressures were measured. Right-to-left shunting through a patent foramen ovale is known as a rare complication after pneumonectomy. Our patient, however, demonstrated a true atrial septal defect (septum secundum defect) upon open operative repair of the interatrial connection.

Aged↗
PubMed1997

t(3;21) following peripheral blood stem cell transplantation in chronic phase chronic myeloid leukaemia.

A 40-year-old male with Ph-positive CML underwent PBSC autografting after initial treatment with hydroxyurea and interferon. Following autograft he remained in chronic phase with cytogenetic or molecular evidence of low levels of residual Ph-positive cells. However, additional cytogenetic abnormalities, including t(3;21) typically seen in therapy-related myelodysplastic syndrome (MDS) and AML and blast crisis of CML, developed as an independent cell line following the autograft. More than 4 years after the autograft, the patient remains in chronic phase with no evidence of accelerated phase or blast crisis of CML, but with a concurrent MDS. We report a case of CML who developed therapy-related MDS following PBSC autograft while still remaining in chronic phase.

Adult↗
PubMed1997

Use of mannose ligands in IVF screens to mimic zona pellucida-induced acrosome reactions and predict fertilization success.

A predictive test for determining whether motile populations of human spermatozoa will fertilize eggs in vitro has been an elusive goal of clinical research. We have developed an assay for the ability of motile human spermatozoa to bind fluorescein isothiocyanate-labelled mannosylated bovine serum albumin (Man-FITC-BSA) as a test for the presence of sperm surface receptors (lectins) for mannose ligands. Mannosylated ligands are present on the human zona pellucida and are involved in the species-specific binding of human spermatozoa to the zona pellucida. We now demonstrate in prospective blinded analysis that the fractional increase in acrosome loss following a mannose ligand challenge is highly correlated with the rate of fertilization in vitro. Using an incremental increase of acrosome exocytosis of >0.1 as a threshold to predict which specimens will yield normal fertilization, the assay has a sensitivity of 97.8%, a specificity of 83.3%, a positive predictive value of 95.7% and a negative predictive value of 90.7%. These data indicate that testing for a mannose-induced acrosome reaction may be useful in assessment of sperm function prior to in-vitro fertilization in order to assign males to conventional insemination or intracytoplasmic sperm injection protocols.

Acrosome↗
PubMed1997

Visual outcome and complications of residents learning phacoemulsification.

The increasing popularity of phacoemulsification in our country raises important training issues. We prospectively analyzed the incidence of complications and visual outcomes in the initial 70 phacoemulsifications (70 patients) performed by the first two residents learning phacoemulsification in our training programme. Both were experienced in standard (manual) extracapsular cataract extraction. Postoperative follow up of 6 weeks or longer was available in 59 eyes. The 11 patients (11 eyes) lost to follow up did not have any intra-operative complications. The overall incidence of vitreous loss was 10%, similar to the frequency of this complication (determined retrospectively) in the first 70 standard extracapsular cataract extractions performed by the same residents. Intraocular lenses (IOL) were successfully implanted in 62 eyes, as planned. One IOL dislocated into the vitreous was successfully repositioned. Other complications encountered included superior corneal edema (3 eyes), iris damage inferiorly (7 eyes) and clinical cystoid macular edema (5 eyes). A best corrected visual acuity of 6/12 or better was obtained in 56 (94.8%) of the 59 eyes available for the six week follow up. In the eyes with vitreous loss, 6 out of 7 had visual acuity better than 6/12. No nuclei were lost into the vitreous. The rate of surgical complications for residents learning phacoemulsification in a supervised manner can be acceptably low.

Clinical Competence↗
PubMed1996

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the detection of polymerase chain reaction products containing 7-deazapurine moieties.

Double-stranded DNA fragments of different length with and without the incorporation of 7-deazapurine deoxynucleotides have been prepared via the polymerase chain reaction (PCR) using exo(-)Pseudococcus furiosus DNA polymerase, unmodified primers, and c7-dATP and c7-dGTP as the only purine triphosphates. In spite of the presence of some unmodified purine moieties due to the primers, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed remarkable differences between the modified and unmodified DNA fragments. The incorporation of 7-deazapurine nucleotides resulted in a significant reduction of fragmentation and therefore in increased signal intensities and higher mass resolution. The molecular weights could be determined with an accuracy of up to 0.03%. Mass resolution was sufficient to resolve the (M + H)+ signals of the two single strands of a 7-deazapurine-containing 99-base-pair DNA duplex. Thus, MALDI-TOF mass spectrometry offers a very fast and accurate way to detect and analyze short PCR products sufficient in length for many diagnostic applications without gel electrophoresis and labeling.

Autoradiography↗
PubMed1996

Analysis of ligase chain reaction products via matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry.

A rapid and accurate detection of ligation products generated in ligase chain reactions (LCR) by using matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) is reported. LCR with Pfu DNA ligase was performed with a wild-type template and a template carrying a single point mutation within the Escherichia coli lacI gene as a model system. Starting from about 1 fmol of template DNA the ligation product generated in the positive reactions was analyzed with HPLC and MALDI-TOF-MS, whereby the need of proper sample purification prior to mass spectrometric analysis was demonstrated. A purification procedure with a high potential for automation using streptavidin-coated magnetic particles and ultrafiltration was introduced. Plasmid DNA and short single-stranded oligonucleotides have been used as template. A point mutation could be discriminated from the wild-type template due to the absence or presence of ligation product. This approach allows the rapid-specific detection of template DNA in femtomole amounts and moreover can distinguish between sequence variations in DNA molecules down to point mutations without the need for labeling, gel electrophoresis, membrane transfer, or hybridization procedures.

Base Sequence↗
PubMed1996

Detection of hepatitis B virus DNA in serum samples via nested PCR and MALDI-TOF mass spectrometry.

In a blind study, nested polymerase chain reaction (PCR) was performed with control DNA and DNA preparations from serum samples of six patients. The detection limit was determined to be 100 molecules of template in 1 ml of serum. Hepatitis B virus (HBV) related products of nested PCR were purified by ultrafiltration and immobilisation on streptavidin coated magnetic beads. The immobilized PCR products were denatured from the beads and analyzed via matrix assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. The results of MALDI-TOF MS analysis were in agreement with the results obtained by polyacrylamide gel electrophoresis (PAGE) and with the data obtained by serological analysis. The detection strategy introduced here has a high potential for automation and represents a fast and reliable method of detection for HBV DNA in serum without the need for time consuming gel electrophoresis and labeling or hybridization procedures.

DNA, Viral↗
PubMed1996

Nonsteric factors dominate binding of nitric oxide, azide, imidazole, cyanide, and fluoride to the rhizobial heme-based oxygen sensor FixL.

BACKGROUND: The FixL protein is a heme-based sensor. Binding of oxygen to a unique heme domain inhibits a kinase domain of the type found in two-component regulators. Oxygen association is slow, but the dissociation rate is comparable to that of myoglobins. We have probed the size and chemistry of the FixL heme pocket by measuring the affinites, on rates and off rates for a wide variety of ferric heme ligands. Cyanide, but not fluoride, regulates the kinase activity. To examine how the sensory heme domain interacts with the kinase, we asked how the presence of the kinase domain affects ligand binding. RESULTS: The affinities of ferric FixL for heme ligands follow the same trend as their pKa values: cyanide > 4-methyl imidazole > imidazole > fluoride > azide >> thiocyanate. The association rates follow the reverse trend. Striking differences from myoglobin include a 6-fold greater affinity for, and faster binding to, the bulky ligand imidazole, a 14-fold faster on rate for nitric oxide, a 2 800-fold lower affinity for azide, and a complete failure to bind thiocyanate. The presence of the kinase domain does not alter the affinity or binding kinetics of the high-spin ligand fluoride, but affects the off rates of other ligands. The EPR spectrum shows a characteristic pentacoordinate nitrosyl heme, indicating that the Fe-His bond in FixL is strained. CONCLUSIONS: The importance of ligand deprotonation to the on rates and the fact that large ligands bind readily indicate that the heme pocket is open and apolar. Ligand basicity strongly influences the strength of binding. The destabilization of inhibitory ligands by the presence of the kinase domain is consistent with a 'load' imposed by coupling to the inactivating mechanism.

Azides↗
PubMed1996

Estradiol attenuates directed migration of vascular smooth muscle cells in vitro.

Although the cardiovascular benefits of the hormone estrogen are at least, in part, mediated by its antiproliferative effect on vascular smooth muscle, its action on the migration of these cells is unknown. To explore this relationship, female rat aortic smooth muscle cells were grown in hormone-free medium, and the effect of various concentrations of beta-estradiol on directed cellular migration was measured in vitro using a microwell Boyden chamber apparatus. Migration of smooth muscle cells to the known chemoattractants platelet-derived growth factor, insulin-like growth factor-1, and fibronectin (all at peak doses for migratory activity) was attenuated by beta-estradiol (0.5 to 10 ng/ml) in a concentration-dependent manner relative to control cells treated with vehicle (0.01% ethanol). This response was insensitive to pretreatment with indomethacin and was stereospecific (17 alpha-estradiol lacked response). Like beta-estradiol, the synthetic estrogen diethylstilbestrol attenuated directed smooth muscle cell migration whereas the male hormone testosterone was ineffective. Additional studies showed that beta-estradiol-mediated suppression of migration was inhibited by the anti-estrogen ICI 164,384 and the gene transcription inhibitor actinomycin D. These are the first results demonstrating a reduction in directed smooth muscle cell migration by beta-estradiol. The mechanism of this estrogen-mediated response appears to involve conventional estrogen receptors.

Animals↗
PubMed1995

Induction of gene expression by intracellular interferon-gamma: abrogation of the species specificity barrier.

We reported previously that murine L-929 cells expressing a human interferon (IFN)-gamma cDNA lacking a signal peptide sequence synthesize but fail to secrete human IFN-gamma and support viral replication at a reduced level. These cells also had elevated levels of IFN-inducible gene products. We show here that a similar response is seen in human cells expressing a mutated murine IFN-gamma cDNA. The ability of human IFN-gamma to induce gene expression in murine cells is shown to be due to the intracellular IFN-gamma rather than to clonal variation, induction of endogenous murine IFN, or alternative mediators of antiviral activity. We have used a murine cell line, Ltk-aprt-, which is resistant to both type I and II IFNs but responsive to combined treatment with both. Ltk-aprt- cells transfected with human IFN-gamma cDNA lacking a signal sequence support virus replication at the same level as control cells. However, unlike transfectants containing only the neoR selection gene, clones expressing the mutated human IFN-gamma gene show strong protection against viral infection and elevated levels of 2,5 A synthetase mRNA and MHC class I protein after treatment with IFN-beta alone. Reverse transcriptase-PCR rules out the induction of endogenous murine IFN expression as a mediator of these effects. Thus, expression of intracellular human IFN-gamma mimics treatment with extracellular murine IFN-gamma in permitting a synergistic response to IFN-beta. Given the inability of human IFN-gamma to bind to the murine cell-surface receptor our results show that intracellular IFN-gamma can activate certain responses independent of cell-surface binding.

Animals↗
PubMed1995

Molecular cloning and expression pattern of genes from a 470 Kb region near BRCA1 locus on chromosome 17q21.

We have used direct cDNA selection to isolate expressed sequences from a set of four overlapping YACs, spanning approximately 470 kb of the chromosomal interval 17q21 centromeric to BRCA1 gene. Thirty-eight nonoverlapping unique cDNA fragments were identified in this region. Twenty-two of the selected cDNAs showed complete identity with known genes and expressed sequence tags. Two of these cDNAs shared sequence homology with genes known to encode potential DNA binding motifs and hence could function in transcriptional regulation. The remaining 16 unique cDNA fragments showed no significant similarity to sequences in the databases and could potentially encode novel genes. Northern analysis of the novel cDNAs showed differential expression in various tissues, supporting the transcribed nature of these sequences. Our results place the gene for a G-protein coupled receptor (GPR2) previously mapped to 17q within a 400 kb region on 17q21.

BRCA1 Protein↗
PubMed1994

Retinoic acid-mediated activation of HNF-3 alpha during EC stem cell differentiation.

We present evidence demonstrating that the liver-enriched transcription factor HNF-3 alpha is activated upon retinoic acid-induced differentiation of mouse F9 embryonal carcinoma cells. We have detected increases in the DNA binding activity and mRNA level of HNF-3 alpha. Both are reflections of the actual activation mechanism at the level of transcriptional initiation, which we showed with the help of HNF-3 alpha promoter constructs. Time course studies clearly show that HNF-3 alpha activation is a transient event. Employing Northern blots, HNF-3 alpha mRNA can be detected between 16 and 24 hours post-differentiation, reaches its zenith at approximately 1 day, and then declines to virtually undetectable levels. F9 cells can give rise to three distinct differentiated cell types; visceral endoderm, parietal endoderm, and primitive endoderm. We have clearly shown that HNF-3 alpha stimulation occurs upon primitive endoderm formation. In addition, the transcription factor is also activated during the induction of cell lineages that give rise to parietal and visceral endoderm. HNF-3 alpha stimulation upon visceral endoderm differentiation is accompanied by the activation of HNF-3 target genes such as transthyretin, suggesting that HNF-3 alpha is involved in the developmental activation of this gene. In contrast, HNF-3 alpha target genes in parietal and primitive endoderm have yet to be identified. However, the stimulation of HNF-3 alpha during primitive endoderm formation, which is an extremely early event during murine embryogenesis, points towards a role for the factor in crucial determination processes that occur early during development.

Animals↗
PubMed1994

Delayed activation of HNF-3 beta upon retinoic acid-induced teratocarcinoma cell differentiation.

We have investigated the retinoic acid-mediated activation of the transcriptional regulator HNF-3 beta during differentiation of mouse F9 embryonal carcinoma cells. Using gel shifts, HNF-3 beta DNA binding activity was clearly detected in differentiated cells, while F9 stem cells were devoid of this activity. We also demonstrated that HNF-3 beta mRNA is specific for differentiated cells. Addition of retinoic acid to F9 stem cells results in delayed activation of HNF-3 beta mRNA which can be detected 1-2 days after the initiation of differentiation. HNF-3 beta mRNA concentrations are maximal at approximately 4 days postdifferentiation and stay at elevated levels for at least 4 additional days. Nuclear run-on experiments clearly show that HNF-3 beta is activated at the level of transcriptional initiation, suggesting that the increases of beta-specific DNA binding activity and mRNA concentration are merely a reflection of this activation mechanism. F9 cells can give rise to three distinct differentiated cell types, visceral endoderm, parietal endoderm, and primitive endoderm, and we have observed HNF-3 beta stimulation during the formation of all three tissues. HNF-3 beta stimulation upon visceral endoderm differentiation is accompanied by the activation of HNF-3 target genes such as transthyretin, suggesting that HNF-3 beta is involved in the developmental activation of this gene. In contrast, HNF-3 beta target genes in parietal and primitive endoderm have yet to be identified. However, the stimulation of HNF-3 beta during primitive endoderm formation, which is an extremely early event during murine embryogenesis, points toward a role for the factor in crucial determination processes that occur early during development.

Animals↗
PubMed1994

Randomized controlled trial of oral captopril, of oral isosorbide mononitrate and of intravenous magnesium sulphate started early in acute myocardial infarction: safety and haemodynamic effects. ISIS-4 (Fourth International Study of Infarct Survival) Pilot Study Investigators.

The purpose of this randomized controlled study was to assess the haemodynamic effects, safety and tolerability in acute myocardial infarction (AMI) of one month of oral captopril, one month of oral isosorbide mononitrate and 24 h of intravenous magnesium. It was carried out in four United Kingdom and six Polish hospitals in consecutive phases: oral captopril vs oral mononitrate vs placebo were compared among 400 patients in a 'three-way' study; and then oral captopril vs placebo and oral mononitrate vs placebo were compared among 474 patients in '2 x 2' and '2 x 2 x 2' factorial studies (with 208 patients in the latter study also randomized between intravenous magnesium and open control). The factorial studies differed from the three-way study in that one group of patients was allocated both oral captopril and oral mononitrate, a higher maintenance dose of captopril was used (following the same initial dose), and once daily controlled-release mononitrate was used. In the three-way study, the mean of the lowest systolic blood pressures recorded during the first 4 h after randomization were (mmHg +/- standard error): 104 +/- 2 captopril vs 105 +/- 1 mononitrate vs 112 +/- 2 placebo (P < 0.001 for captopril or for mononitrate vs placebo), and in the factorial studies were 105 +/- 1 captopril vs 110 +/- 1 placebo (P < 0.01) and 106 +/- 1 mononitrate vs 108 +/- 1 placebo (NS). There was an excess of hypotension recorded among patients allocated active treatment (captopril > mononitrate > placebo) and there was a small, but significant, excess of cardiogenic shock with captopril compared with control in the factorial study. However, in these studies, neither captopril nor mononitrate were associated with any overall increase in the incidence of hypotension considered severe enough to lead to treatment being stopped. No other serious complications were observed, and compliance with study tablets at hospital discharge was not significantly different between the active and placebo groups. Patients allocated magnesium in the 2 x 2 x 2 factorial study had a slightly lower mean systolic blood pressure just after the initial 15 min bolus injection (126 +/- 2 magnesium vs 134 +/- 3 control; P < 0.05) but there were no significant differences during the subsequent 24 h maintenance infusion period. Apart from some facial flushing, magnesium did not appear to be associated with any complications.(ABSTRACT TRUNCATED AT 400 WORDS)

Captopril↗
PubMed1994

Chronic granulocytic leukaemia presenting with an extramedullary T lymphoblastic crisis.

A 33-year-old man presented with hepatosplenomegaly and lymphadenopathy. Bone marrow findings were consistent with Philadelphia-positive accelerated-phase chronic granulocytic leukaemia (CGL). Lymph node histology and immunhistochemistry were diagnostic of T lymphoblastic lymphoma. Molecular studies on lymph node DNA identified rearrangement within the major breakpoint cluster region (M-bcr), indicating a common clonal origin of CGL and the T lymphoblastic lymphoma. We report the second case where extramedullary T lymphoblastic crisis was the presenting feature of CGL.

Adult↗
PubMed1993

Inhibition of viral growth by an alpha-oligonucleotide directed to the splice junction of herpes simplex virus type-1 immediate-early pre-mRNA species 22 and 47.

A 13-residue alpha-anomeric oligonucleotide [alpha-5'-d(GGGCGTCCTCCTT)3'], 5'-substituted with a psoralen derivative, Pso-alpha-13 psoralen linked to the 5' end of an alpha-anomeric n-residue oligonucleotide, was targeted to the acceptor splice junction of Herpes simplex virus type-1 immediate-early pre-mRNA species 22 and 47. Inhibition of viral growth was observed upon irradiation of Vero cells infected with Herpes simplex virus type-1 and treatment with Pso-alpha-13. The virus titer was decreased by 80% at an oligonucleotide concentration of 0.5 microM and at a multiplicity of infection of 0.1 plaque-forming units/cell. The 13-residue oligonucleotide did not induce any cytotoxic effect after irradiation and the inhibition of viral growth was clearly sequence specific. A non-specific 5' Pso-alpha-15 did not inhibit Herpes simplex virus type-1 growth. The 5' Pso-alpha-13 targeted to the acceptor splice junction of Herpes simplex virus type 1, contained five mismatches with respect to the corresponding sequence of Herpes simplex virus type 2, and did not exhibit any inhibitory effects on Herpes simplex virus type-2 growth. These results show that alpha-oligonucleotides can exhibit a sequence-specific antiviral effect and suggest that they may inhibit splicing reactions and be useful in targeting specific nucleic acid sequences within the cell nucleus.

Animals↗