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Biomedical subjects

J Levin

Publications and source records attributed to J Levin.

At least 55 records · Page 3Linked to original sources
PubMed1995

Effects of bacterial endotoxin on human cross-linked and native hemoglobins.

Previous investigations have demonstrated that hemoglobin (Hb) is a binding protein for bacterial endotoxin (lipopolysaccharide, LPS) and that the structure and biological activity of LPS are altered in the presence of Hb. In the present study, the influence of LPS on the structure of native human HbA0 and covalently cross-linked Hb (alpha alpha Hb) was studied by analyzing the absorption and circular dichroic spectra of Hb in the wavelength region of 200-650 nm. Incubation of oxyHb with each of several LPSs resulted in a decrease in the intensity of the major Soret band at 414 nm with a shift in the maximum peak to 410 nm, decreases in the intensities of the major visible region peaks at 541 and 577 nm, and the appearance of increased absorbance in the visible region in the range of 630 nm. The resultant spectra are characteristic of methemoglobin formation. These spectral changes were time-dependent and LPS-concentration-dependent. Production of methemoglobin was prominent with chemically modified, partially deacetylated rough LPS, and was observed to a lesser extent both with native, complete rough and with native smooth LPSs. The influence of LPS on the absorption spectrum of methemoglobin also was directly tested. The conversion of methemoglobin to hemichrome in the presence of LPS was demonstrated and was shown to be reversible. Analysis of circular dichroic spectra of Hb demonstrated LPS-induced spectral changes in the visible and Soret regions consistent with the production of a substantial quantity of metHb, but did not demonstrate any alteration in the far-UV region (210-240 nm). Moreover, Hb oxygen affinity was only slightly altered after incubation with any of several LPSs. In conclusion, analyses of absorption and circular dichroic spectra reveal the potential of LPS to produce a facilitated oxidation of both alpha alpha-cross-linked human Hb and native human HbA0, without substantial changes in the secondary structure of the globin.

Bacterial Toxins↗
PubMed1995

The role of paclitaxel in the treatment of head and neck cancer.

Current chemotherapeutic approaches to recurrent head and neck cancer have routinely yielded response rates of 10% to 30% (for single agents) and 30% to 50% (for combination chemotherapy). However, median survival times for patients with metastatic and/or recurrent disease have stagnated at around 6 months since the 1970s. The investigation of new drugs and treatment approaches thus continues to be a high priority. One such agent, paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ), has shown good single-agent activity and is also believed to potentiate the effects of radiation therapy in patients with head and neck cancer. We have focused on the addition of paclitaxel to the previously established combination of 5-fluorouracil, hydroxyurea, and radiation therapy. The study goals are to establish the maximum tolerated dose and dose-limiting toxicity of paclitaxel when added to this combination as a 5-day continuous infusion on a biweekly schedule. Preliminary results of this ongoing study have demonstrated activity in patients with poor-prognosis head and neck cancer. Major dose-limiting toxicities have consisted of neutropenia and mucositis, and definition of a recommended phase II dose is in progress.

Adult↗
PubMed1995

The effect of cell-free hemoglobin on intravascular clearance and cellular, plasma, and organ distribution of bacterial endotoxin in rabbits.

Cell-free hemoglobin (Hb) and bacterial endotoxin (LPS) synergistically produce toxicity. To elucidate possible mechanisms, three groups of rabbits received LPS alone, LPS plus human serum albumin (HSA), or LPS plus Hb (Hb group). The intravascular retention of injected iodine 125-labeled LPS during the 30-minute period analyzed was significantly longer in the Hb group than in the LPS alone or HSA groups (p = 0.0007 and p = 0.03, respectively), especially during the initial 10 minutes. The intravascular half-life of LPS in the LPS control, HSA, and Hb groups was 2.8, 4.0, and 4.9 minutes, respectively; the area under the curve was 1369 +/- 483, 1594 +/- 360, and 1731 +/- 481, respectively (ng/ml x minutes, mean +/- SD); and the total body clearance was 24.7 +/- 9.2, 20.1 +/- 5.4, and 18.9 +/- 6.0 (ml/min, mean +/- SD), respectively. The proportion of LPS associated with blood cells was very small at the initial 1-minute time period, and this decreased even further during the 30-minute period analyzed (p = 0.0001). Over 96% of injected LPS was associated with the cell-free plasma, with 51% to 54% of LPS in the apoprotein fraction at the initial time point and 35% to 37% in the HDL fraction. The proportion of LPS increased significantly in the HDL fraction and decreased significantly in apoproteins during the 30-minute period analyzed (p = 0.0006 and p = 0.002, respectively). However, there were no differences between the three groups. The liver was the main distribution site (74%) of injected LPS among the six organs evaluated. In the Hb group the accumulation of 125I-labeled LPS in the spleen was significantly lower than that in the HSA group (p = 0.05). The synergism of the in vivo toxicity reported for LPS and Hb may be due, in part, to the decreased rate of intravascular clearance of endotoxin.

Adrenal Glands↗
PubMed1995

An overview of thrombopoietin: with a historical perspective.

A physiologically relevant thrombopoietin (TPO) must be a humoral regulator with lineage specificity for megakaryocytes and their precursors. It should be capable of stimulating platelet production in normal animals, and elevated levels of TPO should be detectable in the plasma following acute, severe thrombocytopenia. Acute thrombocytopenia provides a model system that is likely to predict the effects of TPO, since many of the effects on megakaryocytes and platelets observed after induction of acute thrombocytopenia would be mediated by TPO. Important questions remain to be answered. Do the currently available data for the c-Mpl ligand explain previously published data that describe elevated levels of Meg-CSF in the circulation following production of bone marrow aplasia? Does the c-Mpl ligand account for all of the megakaryocyte stimulatory factors that have been described? Is there another factor that accounts for at least some of the acute alterations in megakaryocytopoiesis that occur immediately following a decrease in platelet levels?

Acute Disease↗
PubMed1994

Hemoglobin, a newly recognized lipopolysaccharide (LPS)-binding protein that enhances LPS biological activity.

Cell-free hemoglobin (Hb) is a purified preparation of human hemoglobin that is being developed as a resuscitation fluid. In vivo administration of hemoglobin has resulted in significant toxicity, due in part to contamination with bacterial endotoxin (lipopolysaccharide (LPS)). To better understand this toxicity, we have studied the interaction between Hb and LPS. Mixtures of each of three different Hb preparations (cross-linked alpha alpha Hb, cross-linked carbon monoxy-alpha alpha HbCO, and non-cross-linked (native) HbAo) and LPS (Escherichia coli O26:B6 or Proteus mirabilis S1959) were examined by several independent methods for evidence of Hb.LPS complex formation. Binding assays in microtiter plates demonstrated saturable binding of LPS to immobilized Hb, with a kD of 3.1 x 10(-8) M. Binding of LPS to Hb also was demonstrated wiht a radiolabeled LPS photoaffinity probe. Ultrafiltration of Hb/LPS mixtures by 300- and 100-kDa cut-off membranes showed that the majority of LPS in these mixtures (87-97 and 64-72%, respectively) was detected in the filtrates, in contrast to the lack of filterability of LPS in the absence of Hb. Density centrifugation demonstrated that LPS co-migrated with each of the three Hbs, whereas unbound LPS had a distinctly greater sedimentation velocity than Hb or Hb.LPS complexes. Nondenaturing polyacrylamide gel electrophoresis demonstrated that in the presence of Hb, LPS migrated into the gel and co-electrophoresed with Hb, whereas LPS alone did not appreciably enter the gel. Finally, precipitation by ethanol of each of the three Hb preparations was increased in the presence of LPS compared with precipitation in the absence of LPS. Interaction of LPS with each of the three Hb preparations was also associated with altered biological activity of LPS, as shown by enhancement of LPS activation of Limulus amebocyte lysate. Therefore, our data provide several lines of independent evidence for Hb-LPS complex formation and indicated that LPS exhibited altered physical characteristics and enhanced biological activity in the presence of Hb.

Acute-Phase Proteins↗
PubMed1994

Immunocytochemical identification of murine and human megakaryocyte colonies in soft-agar cultures.

Murine megakaryocyte (MK) colonies in soft-agar cultures were immunocytochemically stained with platelet antiserum and an immuno-alkaline phosphatase procedure. Subsequently, cytochemical staining for acetylcholinesterase was used to confirm the specificity of the immunolabelling technique. The correlation of numbers of megakaryocyte colonies enumerated by independent observers was excellent. A comparable platelet antiserum directed against human platelet epitopes was utilized to identify human MK colonies in soft-agar cultures of human bone marrow. Using this method, we determined that the frequency of detectable human MK colonies in our agar culture system was maximal between days 10 and 12. The immunocytochemical staining technique we have developed for identification of MK colonies in soft-agar cultures yielded good cellular morphology and produced an intensely specific label against a clear background; it therefore facilitated accurate enumeration of MK colonies. This non-fluorescent method avoids dependence upon a non-permanent marker, and allows the simultaneous enumeration of positive and negative colonies.

Acetylcholinesterase↗
PubMed1994

Hemoglobin: a newly recognized binding protein for bacterial endotoxins (LPS).

Administration of purified hemoglobin (Hb) as a cell-free resuscitation fluid is associated with multiple organ toxicities. Many of these toxicities are characteristic of the pathophysiological effects of bacterial endotoxins (lipopolysaccharide, LPS). To better understand the potential role of LPS in the observed in vivo toxicities of Hb, we examined mixtures of Hb and LPS for evidence of LPS-Hb complex formation. LPS-Hb complexes were demonstrated by three techniques: ultrafiltration through 300 kDa cut-off membranes, which distinguished LPS in complexes (87-89% < 300 kDa) from LPS alone (90% > 300 kDa); density centrifugation through sucrose, which distinguished denser LPS alone from LPS-Hb complexes; and precipitation by 67% ethanol, which demonstrated 2-3 fold increased precipitability of Hb in complexes compared to Hb alone. Interaction of LPS with Hb was also associated with markedly increased biological activity of LPS, as manifested by enhancement of LPS activation of Limulus amebocyte lysate (LAL), increased release of human mononuclear cell tissue factor, and enhanced production of human endothelial cell tissue factor. These results demonstrated that hemoglobin can serve as an endotoxin binding protein, and that this interaction results in the alteration of several of the physical characteristics of LPS and enhancement of the biological activities of LPS. These findings suggest that a mechanism for the toxicity of infused Hb in vivo may involve potentiation of the biological effects of LPS. In addition, these observations suggest a mechanism by which LPS-related morbidity during sepsis could be enhanced by erythrocyte hemolysis.

Acute-Phase Proteins↗
PubMed1993

A single amino-acid substitution in the Ets domain alters core DNA binding specificity of Ets1 to that of the related transcription factors Elf1 and E74.

Ets proteins form a family of sequence specific DNA binding proteins which bind DNA through a 85 aminoacids conserved domain, the Ets domain, whose sequence is unrelated to any other characterized DNA binding domain. Unlike all other known Ets proteins, which bind specific DNA sequences centered over either GGAA or GGAT core motifs, E74 and Elf1 selectively bind to GGAA corecontaining sites. Elf1 and E74 differ from other Ets proteins in three residues located in an otherwise highly conserved region of the Ets domain, referred to as conserved region III (CRIII). We show that a restricted selectivity for GGAA core-containing sites could be conferred to Ets1 upon changing a single lysine residue within CRIII to the threonine found in Elf1 and E74 at this position. Conversely, the reciprocal mutation in Elf1 confers to this protein the ability to bind to GGAT core containing EBS. This, together with the fact that mutation of two invariant arginine residues in CRIII abolishes DNA binding, indicates that CRIII plays a key role in Ets domain recognition of the GGAA/T core motif and lead us to discuss a model of Ets proteins--core motif interaction.

Amino Acid Sequence↗
PubMed1993

Induction of apoptosis in blood cells from a patient with acute myelogenous leukemia by SC41661A, a selective inhibitor of 5-lipoxygenase.

Participation of leukotriene products in normal ex vivo hematopoiesis is well established. With increasingly specific inhibitors of lipoxygenases, it becomes possible to more closely define any participation of their biosynthetic products in these events. We cultured chronic myelogenous leukemia cells from the peripheral blood of several patients in blast crisis with three inhibitors of lipoxygenases: ETYA, and the more selective A63162 (Abbott) or SC41661A (Searle). All three agents reduced labelling of DNA with H3 thymidine measured at 4 h and reduced cell numbers by 72 h. An antisense deoxyoligonucleotide to the 5-lipoxygenase mRNA 'start' codon inhibited DNA synthesis at 24 h, as did two control oligonucleotides. Marked nuclear ultrastructural changes characteristic of apoptosis were induced by SC41661A in a subset of cells with the ultrastructure of promyelocytes. Whether this response characterizes a common pattern of this subset of leukemic cells to SC41661A, if damage to mitochondria with reduced function of bcl-2 protooncogene product located at that site might have contributed or some other mechanism was responsible, and if inhibition of 5-lipoxygenase activity was involved, are questions to be decided in the future.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1993

Failure of the International Normalized Ratio to generate consistent results within a local medical community.

Use of the International Normalized Ratio (INR) has been recommended as a means of standardizing prothrombin time (PT) results for management of oral anticoagulant therapy. During the evaluation of a new lot of thromboplastin reagent, however, INR values were obtained that were inconsistent with results obtained with the prior lot of reagent from the same manufacturer. A local normalized ratio (LNR) was substituted for the INR for the new reagent, based on a calculated local sensitivity index (LSI). Validation of the LSI was performed at the three hospitals in the medical community by testing an identical panel of aliquots from 64 plasmas obtained from patients who were chronically receiving oral anticoagulants. Each test result was classified according to the INR value as low, low therapeutic, high therapeutic, or high. Local normalized ratio values obtained at one hospital in the community were in reasonable agreement with INR determinations in the other two hospital laboratories. Classification mismatches occurred using the INR, however, in 84 of 280 (30%) of the paired samples. Thus, the inability to generate consistent INR values within a local medical community raises serious concern about the reliability of the INR concept in its current form.

Anticoagulants↗
PubMed1993

A reevaluation of the role of crystalloid preload in the prevention of hypotension associated with spinal anesthesia for elective cesarean section.

BACKGROUND: Hypotension after spinal anesthesia for cesarean section remains a common and serious complication despite the use of uterine displacement and volume preloading. The current study revaluated the role of crystalloid volume preloading in this context. METHODS: In a two-stage open sequential design, patients presenting for elective repeat cesarean section were allocated to receive either no preload or 20 ml/kg crystalloid administered over 15-20 min before spinal anesthesia. Hypotension was defined as a decrease in systolic pressure to less than 100 mmHg and to less than 80% of baseline value, and the study was designed to detect a 20% difference in the incidence of hypotension between the groups, with statistical significance at the 10% (alpha = 0.1) level, one-tailed. RESULTS: One hundred forty patients were studied. Hypotension occurred in 43 (55%, 95% CI 43.4-66.4) preloaded and 44 (71%, 95% CI 58-81.8) unpreloaded subjects, a difference in incidence of 16% (95% CI 0.04-31.6), which was statistically significant. There were no significant differences in the severity, timing, or duration of hypotension; the dose requirement for ephedrine; or the clinical and biochemical status of neonates between the groups. The only difference seen was a lower mean base excess (-3.4, SD 2.81 mM-1) in the neonates of hypotensive mothers compared to neonates of nonhypotensive mothers (-2.4, SD 1.99 mM-1). CONCLUSIONS: The study confirms that hypotension associated with spinal anesthesia for cesarean section cannot be eliminated by volume preloading in the supine wedged patient. The relatively small reduction in incidence of hypotension challenges our perception of the value of crystalloid preload. Though volume preload in the elective cesarean section is advocated, the requirement for a mandatory administration of a fixed volume before spinal anesthesia for urgent cases has been abandoned.

Adult↗
PubMed1993

Distribution of bacterial endotoxin in human and rabbit blood and effects of stroma-free hemoglobin.

Bacterial endotoxin (lipopolysaccharide [LPS]) is known to interact with numerous components of blood, including erythrocytes, mononuclear cells, platelets, neutrophils, lipoproteins, and plasma proteins. The relative affinities of LPS for these elements, and the distribution of LPS between them, are unknown. Cross-linked stroma-free hemoglobin (SFH), a potential substitute for erythrocyte transfusion, produces in vivo toxicity in animals consistent with significant LPS contamination. Therefore, we studied the distribution of LPS in human and rabbit blood and examined whether the presence of SFH altered LPS distribution. In either the presence or absence of SFH, LPS was associated predominantly with high-density lipoproteins and apoproteins. There was lesser binding to low- and very-low-density lipoproteins. Examination of the apoprotein pool by column chromatography and density centrifugation demonstrated that LPS in this fraction was predominantly protein bound. Binding of LPS to SFH resulted in dissociation of a portion of the LPS into low-molecular-weight complexes. Cell-bound LPS was only 2 to 16% of the total and was unaffected by SFH. The distribution among blood cells demonstrated predominant binding to platelets in human blood but predominant binding to erythrocytes in rabbit blood. Cellular distribution was not significantly altered by SFH.

Acute-Phase Proteins↗
PubMed1993

Biological and biochemical characteristics of murine megakaryoblastic cell line L8057.

Biological and biochemical characteristics of a murine megakaryoblastic cell line (L8057), derived from an irradiated C3H/He mouse, were studied. The L8057 cells grew in liquid cultures with a doubling time of 24 to 27 hours. By light and electron microscopy, the L8057 cells were small and basophilic with a high nuclear/cytoplasmic ratio, but large cells (approximately 25 to 60 microns) were sometimes seen. Western blot analysis of cell extracts demonstrated that murine megakaryocyte and platelet antigens, glycoprotein (GP) IIb/IIIa, fibronectin, and factor VIII:RAg were expressed in L8057 cells. Acetylcholinesterase (AChE)-positive cells were rare (< 2.0%). Flow cytometric analysis demonstrated that 8.7% of the cells were spontaneously polyploid (> 4N DNA levels). In response to phorbol diester (TPA, 50 nm), L8057 cells showed a decrease in proliferation and a marked increase in cell size and amount of cytoplasm. The morphology of the cells became similar to that of megakaryocytes, with larger and lobulated nuclei and expansion of the demarcation membrane system compartment. The proportion of cells with ploidy levels greater than 4N increased to 40.8% (8N, 21.3 +/- 3.2%; 16N, 13.1 +/- 2.6%; > or = 32N, 6.4 +/- 4.9% [n = 4]). More than 90% of the cells became AChE-positive. TPA also increased the expression of GP IIb/IIIa, fibronectin and factor VIII:RAg. Thus, the L8057 cell line provides a potentially superior model with which to study the characteristics of megakaryocytic biology, differentiation and biochemistry.

Acetylcholinesterase↗
PubMed1992

Purification of Limulus polyphemus proclotting enzyme.

Horseshoe crabs (Limulus polyphemus and Tachypleus tridentatus) possess a proteolytic blood coagulation system within their amebocytes that, after release and endotoxin activation, generates a polymerized insoluble coagulin clot. Clotting enzyme from horseshoe crab amebocyte lysate is the protease that activates the clottable protein (coagulogen) which then forms the coagulin clot. Comparison of the previously published descriptions of this enzyme has revealed significantly discordant biochemical characteristics. We purified a 60-kDa proclotting enzyme from L. polyphemus amebocyte lysate to a single band on polyacrylamide gel electrophoresis. After electrophoresis, evaluation of enzymatic activity of this protein within gels demonstrated that the band of purified protein corresponded to enzymatic activity, as detected by amidolytic activity for chromogenic substrates and by gelation of coagulogen applied to the gel. The enzymatic activity was inhibited by serine protease inhibitors. The purified proclotting enzyme had a molecular weight and amino acid composition different from the previously published characterizations of proclotting enzymes from both L. polyphemus and T. tridentatus.

Amino Acids↗