Biomedical subjects
Publications and source records attributed to S Patel.
Thirty-six cases of hepatic toxicity associated with felbamate therapy have been collected by the Food and Drug Administration. Five patients died. We describe a case of massive acute hepatic necrosis and death within 40 days of initiation of felbamate therapy for a generalized tonic-clonic seizure disorder. We describe the clinical and histopathologic features.
OBJECTIVE: To compare the safety and efficacy of percutaneous dilational tracheostomy (PDT) with surgical tracheostomy (ST). DESIGN: Prospective randomized trial. SETTING: Public urban teaching hospital. PATIENTS: Twenty-six patients were randomized to undergo PDT and 27 patients to ST. RESULTS: The time from randomization into the study until tracheostomy was performed was 28.5 +/- 27.9 h in the PDT group and 100.4 +/- 95.0 h in the ST group (p < 0.001). PDT was performed in 8.2 +/- 4.9 min vs 33.9 +/- 14.0 min for ST (p < 0.0001). There was no significant difference in intraprocedural complications between the groups. Postprocedural complication rates were 12% for PDT and 41% for ST (p = 0.008). CONCLUSION: PDT is superior to ST logistically. PDT can be performed at the bedside eliminating the risk of patient transport. Because operating room scheduling is not necessary, PDT can be performed earlier once the decision to do a tracheostomy is made, which will improve ICU utilization. PDT is a faster procedure to perform and has fewer postprocedural complications.
The complete sequence of the human gene encoding the 54kDa subunit of the signal recognition particle has been isolated from a cDNA library. Degenerate oligonucleotides based on the 5' and 3' coding region of the canine gene and a mammalian codon usage table, were used to amplify the sequence using PCR. The nucleotide sequence of the human gene shows that the human sequence shares a 95.8% nucleotide sequence homology and 100% amino acid sequence homology to it's canine counterpart. The sequence has been given the accession number X86373 in the EMBL database.
The inversion(16)(p13;q22) gives rise to chimeric transcripts CBFB-MYH11. To date however, no reports have described the full length coding sequence cloned from patient material or the protein product derived from transcripts. We describe here the cloning and sequencing of the coding region of the fusion gene (type A) from patient cells. The sequence is identical to the included portions of the normal constituent transcripts. We report the study of CBFB and CBFB-MYH11 protein using two anti-CBFB antisera. Twenty-two cases of inv(16) leukemia and a number of other cases of AML were examined. The predicted 70 kDa type A or 95 kDa type D CBFB-MYH11 peptide was detected in 20/22 cases of inv(16) AML. CBFB was expressed as a 21 kDa protein in all samples studied, including hematopoietic cell lines of all major lineages.
A novel method for testing the corneal nerve function, through non-invasive measurement of corneal sensitivity, is presented. The method of using a controlled pulse of air to stimulate the corneal surface is described. Technical information of the aesthesiometer's construction is given. Examples of the ability of the instrument to accurately locate the corneal sensitivity threshold using either a method of constant stimuli, or a method of limits, is described for 14 normal, non-lens wearing eyes. Measurements are made in millibars of air pressure required. Mean normal values found were 0.342 +/- 0.068 millibars. Discussion is made of the instrument's mode of action, its advantages over other invasive instruments, and potential clinical application.
We have developed a procedure to measure the rates of apolipoprotein (apoB) and triglyceride secretion from the liver of an individual mouse. Using the well-characterized method of Triton WR-1339 injection to block peripheral removal of newly secreted VLDL, the rate of triglyceride accumulation is monitored and at the end of the experimental period, blood is extracted for quantitative VLDL preparation. ApoB species in isolated VLDL are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the mass of the apoB-48 and apoB-100 species are estimated by Coomassie staining and laser densitometric scanning, using known quantities of LDL-apoB-100 as standards. This methodology was applied to measure the rate of apoB secretion in male and female FVB/N mice and we found that the molar ratio of newly secreted ApoB-48 to B-100 is 4.6 in the male, and 3.8 in the female. Measurements of the steady state apoB levels indicate that liver-derived apoB-48 is cleared from the circulation 7.1 times faster than B-100 in the male and 4.7 times faster in the female mouse. VLDL apoB-48 fractional turnover is approximately 1800 pools per day in both the male and female mouse (1814 +/- 139 vs. 1831 +/- 365 respectively, P = 0.92). ApoB-100 fractional turnover rates are much slower and show a statistically significant difference between males and females (255 +/- 19 pools per day vs. 386 +/- 65 pools per day, respectively, P = 0.006). This procedure provides for quantification of secretory rates of these apo proteins in vivo, and may be useful for studying the effects of genetic manipulation on the simultaneous secretion of apoB-48- and apoB-100-containing VLDL, afforded by the panoply of transgenic mouse models now available for study, as well as for effects of diet and drug therapy.
A method for the laboratory production of a phenol red impregnated thread is described. Using the thread, tear flow was measured in the right eye of 2 groups of normal subjects: group I, n = 20, average age 26.2 years (+/- 5.6); and group II, n = 20, average age 56.4 years (+/- 13.8). Data were collected by placing a thread over the lower lid margin for a time period of 15 s. A further seven threads were inserted at 5 min intervals for periods increasing by 15 s up to 120 s. A significant difference in mean thread wetting between the two age groups was found for thread insertion times of 30, 60 and 90s. For each eye, the rate of thread wetting was exponentially related to the period of thread insertion. Thread wetting and the constant of the exponential were not found to be gender dependent. Simulating the tears in the lower fornix, using a bath of saline, it was concluded that the exponential constant was a property of thread material and not tear fluid. For practical purposes, the rate of thread wetting reached equilibrium by 120 s where mean total thread wetting was 19.5 mm (+/- 6.5). The typical flow of tears y in mm/s, along the phenol red thread can be described by: y = 0.16 + 0.47e-0.0283t where t is the period of thread insertion.
Retinoids down-regulate the expression of metalloproteinases, cytokines, and other genes involved in cell proliferation and inflammation. Tazarotene (AGN 190168), a retinoic acid receptor (RAR)-specific retinoid, is effective in the treatment of psoriasis, a hyperproliferative and inflammatory skin disease. Because negative regulation of genes appears to be important in the antiproliferative and antiinflammatory action of retinoids, we studied the down-regulation of genes in skin raft cultures by this antipsoriatic retinoid. By subtraction hybridization, we found that migration inhibitory factor-related protein (MRP-8) and skin-derived anti-leukoproteinase (SKALP) are down-regulated by AGN 190168. MRP-8 and SKALP are overexpressed in psoriatic lesions as compared to the normal epidermis, and they are markers of hyperproliferative keratinocyte differentiation. We also show that MRP-8 expression is retinoid inhibitable in cultured keratinocytes induced to differentiate with 10% serum or IFN-gamma, and that MRP-8 is inhibited by RAR but not by retinoid X receptor-specific retinoids in a dose-dependent manner. Finally, MRP-8, SKALP, and the previously characterized differentiation marker, transglutaminase I, are all down-regulated in vivo in psoriatic lesions after treatment with AGN 190168 in comparison to placebo. Taken together, these data suggest that these markers may be down-regulated by tazarotene in psoriasis through direct action on keratinocyte gene expression rather than by an overall tazarotene effect on lesional therapeutic status.
We identified a novel azaindole derivative, L-750,667, that has high affinity (Ki = 0.51 nM) and >2000-fold selectivity for D4 dopamine receptors compared with its activity at D2 and D3 dopamine receptors. L-750,667 had little affinity for rat D1/D5 dopamine receptors, sigma binding sites, or 5-hydroxytryptamine1A or 5-hydroxytryptamine2 receptors. In functional studies, L-750,667 exhibited high affinity antagonist activity at D4 receptors, reversing dopamine (1 microM)-induced inhibition of cAMP accumulation in human embryonic kidney (HEK) cells expressing the human D4 receptor (hD4 HEK) with an EC50 value of 80 nM. The radioiodinated form of L-750,667 bound specifically to the human dopamine D4 receptor expressed in HEK cells and saturation analysis revealed a single high affinity binding site for [125I]L-750,667 (Kd = 0.16 +/- 0.06 nM). The maximum number of binding sites (Bmax) estimated using [125I]L-750,667 in hD4 HEK cells was 251 +/- 71 fmol/mg, which correlated well with the Bmax value determined using [3H]spiperone (227 +/- 83 fmol/mg) in the same membrane preparations. The pharmacological profile of [125I]L-750,667 binding to hD4 HEK cells was evaluated using known dopamine receptor agonists and antagonists. The rank order of potencies for dopamine receptor agonists was dopamine > quinpirole > 6,7-aminodihydroxytetralin > 5,6-aminodihydroxytetralin. Dopamine receptor antagonists also showed high affinity, with a rank order of haloperidol > chlorpromazine > domperidone > (+)-butaclamol > (-)-sulpiride = (+)-sulpiride > (+)-SCH23390 > (-)-butaclamol. [125I]L-750,667, bound to D4 receptors in a stereoselective manner with (+)-butaclamol showing higher activity than its respective enantiomer (-)-butaclamol. These results show that [125I]L-750,667 is a novel, highly selective radioligand for dopamine D4 receptors and may be used to investigate the dopamine D4 receptor population in the central nervous system.
The low density lipoprotein (LDL) receptor is part of a family of proteins that mediate the uptake of lipoproteins into cells. In this paper we have demonstrated the over-expression in E. coli of a rat LDL receptor fusion protein that contains the region of the receptor sharing homology with the EGF precursor. The fusion protein was utilized to immunize rabbits and successfully generate antibodies that recognize the intact LDL receptor. These anti-LDL receptor/fusion protein antibodies were used to examine the effects of cyclic AMP on the expression of LDL receptors in isolated rat adipocytes. Incubation of adipocytes with isoproterenol caused a dose-dependent diminution in intact LDL receptors in the plasma membrane with the concomitant appearance of smaller immunoreactive proteins. Pulse-chase experiments demonstrated that isoproterenol rapidly shortened the initial half-life of intact, immunoprecipitable LDL receptors in the plasma membrane. The effects of isoproterenol on LDL receptor expression were mimicked by forskolin, by an analog of cyclic AMP, and by ACTH. In contrast, incubation with propranolol blocked the effects of isoproterenol on LDL receptor expression. While antioxidants and several different protease inhibitors had no effects, N-acetyl-leucine-leucine-methionine (ALLM) was able to prevent the isoproterenol-induced effects on LDL receptors. Thus, it appears that agents acting via cyclic AMP cause a rapid decrease in LDL receptors in the plasma membranes of isolated adipose cells due to the apparent stimulation of an ALLM-sensitive protease that degrades the LDL receptor. These results suggest a novel mechanism for the posttranscriptional regulation of LDL receptor expression in adipocytes.
Increasing phosphorylation reactions by protein kinase A (PKA) or reducing dephosphorylation reactions of protein phosphatase-2A (PP-2A) increases the invasiveness of Lewis lung carcinoma (LLC) cells, as measured by their capacity to traverse extracellular matrix (ECM)-coated filters. Metastatic LLC-LN7 variants have reduced PP-2A activity when compared to nonmetastatic LLC-C8 variants. Immunoblotting showed that this reduced level of PP-2A activity was not due to reduced levels of the PP-2A catalytic (C) subunit. The cellular PP-2A activity could be stimulated by addition of C2-ceramide to LLC-LN7 lysates, or by incubating cells with either C2-ceramide or with a noncalcemic analog of vitamin D3, which has previously been shown to stimulate the release of ceramide. These treatments to elevate PP-2A activity in metastatic LLC-LN7 cells resulted in a decline in their capacity to invade through select (ECM) components, particularly through vitronectin and laminin. Underscoring the importance of PP-2A in limiting the invasiveness of tumor cells was the demonstration that LLC-LN7 cell transfectants overexpressing the PP-2A C alpha subunit were less invasive through ECM components than the wild-type cells. Invasion by these cells was further reduced by additionally increasing PP-2A activity by incubation with C2-ceramide or the vitamin D3 analog. These results suggest a role of a vitamin D3/ceramide/PP-2A pathway in limiting the invasiveness of tumor cells through select ECM components.
Submaximal concentrations of inositol 1,4,5-trisphosphate (InsP3) rapidly release only a fraction of the InsP3-sensitive intracellular Ca2+ stores, despite the ability of further increases in InsP3 concentration to evoke further Ca2+ release. The mechanisms underlying such quantal Ca2+ mobilization are not understood, but have been proposed to involve regulatory effects of cytosolic Ca2+ on InsP3 receptors. By examining complete concentration-effect relationships for InsP3-stimulated 45Ca2+ efflux from the intracellular stores of permeabilized hepatocytes, we demonstrate that, at 37 degrees C, responses to InsP3 are quantal in Ca(2+)-free media heavily buffered with either EGTA or BAPTA [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid]. Lower concentrations of InsP3 were used to examine the kinetics of Ca2+ mobilization at 2 degrees C, because at the lower temperature the stores were more sensitive to InsP3: the concentration of InsP3 causing half-maximal Ca2+ release (EC50) after a 30 s incubation decreased from 281 +/- 37 nM at 37 degrees C to 68 +/- 3 nM at 2 degrees C. At 2 degrees C, the EC50 for InsP3-stimulated Ca2+ mobilization decreased as the duration of exposure to InsP3 was increased: the EC50 was 68 +/- 3 nM after 30 s, and 29 +/- 2 nM after 420 s. InsP3-stimulated Ca2+ mobilization is therefore non-quantal at 2 degrees C: InsP3 concentration determines the rate, but not the extent, of Ca2+ release. By initiating quantal responses to InsP3 at 37 degrees C and then simultaneously diluting and chilling cells to 2 degrees C, we demonstrated that the changes that underlie quantal responses do not rapidly reverse at 2 degrees C. At both 37 degrees C and 2 degrees C, modest increases in cytosolic Ca2+ increased the sensitivity of the stores to InsP3, whereas further increases were inhibitory; both Ca2+ effects persisted after prior removal of ATP. We conclude that the effects of Ca2+ on InsP3 receptors are unlikely either to be enzyme-mediated or to underlie the quantal pattern of Ca2+ release evoked by InsP3.
PIXY321 is a novel fusion protein of recombinant human granulocyte-macrophage colony-stimulating factor and interleukin-3 that exhibits biologic effects of both its parent cytokines in vitro and in preclinical studies. To evaluate the clinical safety and hematopoietic effects of this hybrid cytokine, PIXY321 was administered by subcutaneous injection twice daily at doses of 25 to 1,000 micrograms/m2/day over 14 days to 24 patients with sarcoma before chemotherapy as part of a phase I trial. The treatment was associated with significant increases in white blood cell, neutrophil, platelet, and reticulocyte counts (all P < .001). The increase in neutrophil count was dose-related and was seen during treatment with the cytokine, whereas the increase in platelet count was gradual and peaked after the cessation of the cytokine treatment and was not clearly dose related. PIXY321 treatment also increased bone marrow (BM) cellularity and the percentage of BM cells in S phase (P < .001). In addition, there was a significant increase in the number of CD34+ cells and committed and multipotential progenitors in the peripheral blood. The ex vivo expansion capacity of peripheral blood and BM progenitor cells was preserved after the in vivo treatment with PIXY321. The treatment was well tolerated, with the most common side-effect being injection site reactions. The results of this study show the biologic and clinical activity of a genetically engineered fusion molecule of two hematopoietic cytokines in humans with normal hematopoietic function.
Nine different water soluble polymers reported to strongly affect the properties and structure of water were evaluated for their use in crystallizing a series of 24 different proteins, viruses, and conventional small molecules. All of the polymers produced crystals of some of the molecules and viruses tested, and of the 24 molecules tested, 14 were crystallized. In a number of cases, crystals of the molecules and viruses were obtained under very different conditions than were ever previously used. Because the selection of polymers employed here represents only a sampling of those available to experimenters, we conclude that the potential range of such polymers useful in macromolecular and small molecule crystallization may be very broad.
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OBJECTIVE: To assess the use of quantitative bone scanning (QBS) in the monitoring of patients with intravenous pamidronate-treated symptomatic monostotic Paget's disease of bone in whom biochemical markers of bone turnover are relatively normal. METHODS: QBS was performed in 9 patients and the results were expressed as a ratio, obtained by comparing isotope uptake at an affected and a control (unaffected) site. RESULTS: Serum alkaline phosphatase levels were normal in 7 of the 9 patients and changed minimally with treatment. The median QBS ratio was 2.72 (range 1.69-24.6) at baseline and 1.49 (range 0.63-4.18) posttreatment (P = 0.008). The median symptom score decreased with treatment, but QBS ratios provided the only objective measure of disease activity by which response to pamidronate therapy could be judged. CONCLUSION: QBS may be a useful technique for evaluating the effects of treatment in patients with Paget's disease of bone.
Reliable analytical procedures and certified reference materials are essential for the establishment and enforcement of tolerance levels for ochratoxin A in foods. The inadequacy of analytical procedures, together with the need for certified reference materials, led the Commission of the European Communities Community Bureau of Reference (BCR) to undertake a project to prepare suitable reference materials for ochratoxin A in wheat, in order to improve methodology and to harmonise agreement of results between member states. The first intercomparison study indicated problems in the analysis due to the influence of co-extractives in the matrix, and demonstrated that further work was necessary to improve recovery, clean-up and reproducibility. The second intercomparison study, in the EC Measurements and Testing Programme, correlated the performance of the different methods for ochratoxin A measurement in a separate batch of contaminated wheat, and compared novel immunoaffinity column methods with the standard laboratory methods. Results were obtained from 26 laboratories within 11 European countries, which therefore gives a good representation of the scope of methods currently used in Europe. Considerable improvements in the determination of ochratoxin A were noted compared with the first intercomparison study.
A parametric phase delay estimation technique is used to determine the spatial and inhaled gas composition dependencies of sound propagation time through an intact human lung at frequencies of 150-1200 Hz. Noise transmission measurements from the mouth to the extrathoracic trachea and six sites on the posterior chest wall are performed in 11 healthy adult subjects at resting lung volume after equilibration with air, an 80% helium-20% oxygen mixture, and an 80% sulfurhexafluoride-20% oxygen mixture. The phase delay, tau(f), exhibits a bilateral asymmetry with relatively decreased delays to the left posterior chest as compared with the right. The phase delay to lower lung sites is greater than to upper sites at frequencies below 300 Hz; yet the opposite is found at higher frequencies, indicating changing propagation pathways with frequency. There is no measurable effect of inhaled gas composition on tau(f) below 300 Hz. At higher frequencies, changes in tau(f) that reflect the relative sound speed of the particular inhaled gas are observed. These findings support and extend previous measurements and hypotheses concerning the strong frequency dependence of the acoustical properties of the intact respiratory system.