Critical pathways : a review. Committee on Acute Cardiac Care, Council on Clinical Cardiology, American Heart Association.
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Cardiovascular Diseases↗
Biomedical subjects
J Hochman
Publications and source records attributed to J Hochman.
A disulfide conjugate between anti-tetanus antibodies and HIV (37-72)Tat neutralizes tetanus toxin inside chromaffin cells.
Conjugates between anti-tetanus F(ab')2 fragments and the (37-72) fragment of the HIV Tat protein were taken up by chromaffin cells, NG108-15 neurohybridoma cells and Rev-2-T-6 lymphoma cells. The uptake could not be inhibited by competition with (37-72)Tat, but was reduced in the presence of metabolic inhibitors or at low temperature. The disulfide as well as the thioether conjugate were translocated to the cytoplasmic space, but only the disulfide conjugate moderately restored the stimulated transmitter release inhibited by tetanus toxin. Therefore, disulfide conjugates are more promising than thioethers for the neutralization of intracellular antigens. These conjugates provide new tools to study neuroprotection against bacterial neurotoxins.
Animals↗
Thrombogenic factors and recurrent coronary events.
BACKGROUND: Thrombosis is a pivotal event in the pathogenesis of coronary disease. We hypothesized that the presence of blood factors that reflect enhanced thrombogenic activity would be associated with an increased risk of recurrent coronary events during long-term follow-up of patients who have recovered from myocardial infarction. METHODS AND RESULTS: We prospectively enrolled 1045 patients 2 months after an index myocardial infarction. Baseline thrombogenic blood tests included 6 hemostatic variables (D-dimer, fibrinogen, factor VII, factor VIIa, von Willebrand factor, and plasminogen activator inhibitor-1), 7 lipid factors [cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, lipoprotein(a), apolipoprotein (apo)A-I, and apoB], and insulin. Patients were followed up for an average of 26 months, with the primary end point being coronary death or nonfatal myocardial infarction, whichever occurred first. The hemostatic, lipid, and insulin parameters were dichotomized into their top and the lower 3 risk quartiles and evaluated for entry into a Cox survivorship model. High levels of D-dimer (hazard ratio, 2.43; 95% CI, 1.49, 3.97) and apoB (hazard ratio, 1.82; 95% CI, 1.10, 3.00) and low levels of apoA-I (hazard ratio, 1.84; 95% CI, 1.10, 3.08) were independently associated with recurrent coronary events in the Cox model after adjustment for 6 relevant clinical covariates. CONCLUSIONS: Our findings indicate that a procoagulant state, as reflected in elevated levels of D-dimer, and disordered lipid transport, as indicated by low apoA-1 and high apoB levels, contribute independently to recurrent coronary events in postinfarction patients.
Adult↗
Translocation of acylated pardaxin into cells.
Acylated pardaxin is translocated through the cytoplasmic membrane and is accumulated in the nucleoli of NG108-15 and chromaffin cells. The uptake is time- and dose-dependent and temperature-sensitive. However, the binding of acylated 125I-pardaxin cannot be reduced by competition with pardaxin acylated with Rudinger's reagent. In this respect, acylated pardaxin resembles the Tat protein 37-71 fragment. Metabolic inhibitors do not significantly reduce the uptake of acylated 125I-pardaxin. Acylated pardaxin might be useful as a vector to translocate other molecules.
Acylation↗
Isolation, characterization and synthesis of a novel paradaxin isoform.
We report the isolation of a novel pardaxin isoform from the toxic secretion of the Red Sea Moses sole (Pardachirus marmoratus). Mass spectrometrical analysis of the newly purified peptide revealed a different primary structure compared to the previously known pardaxin isoforms. Sequence analysis disclosed an aspartic acid residue instead of glycine at position 31 of the new isoform. According to the novel sequence, a synthetic Asp-31-peptide was compared with the native compound as well as with synthetic Gly-31-pardaxin. The isolated Asp-31-pardaxin isoform and its synthetic analog exhibited identical elution properties during reverse-phase HPLC, as well as similar dose-dependent lytic effects on human erythrocytes at a concentration of 10(-6) to 10(-5)M. The hemolytic activity of Asp-31-pardaxins was lower than that of Gly-31-pardaxin and no synergistic effect between these peptides was found. The additional negative charge introduced by Asp-31 is likely to affect the selectivity of pardaxin pores towards a variety of ions.
Amino Acid Sequence↗
Nucleolar localization of mouse mammary tumor virus proteins in T-cell lymphomas.
To characterize novel proteins expressed in lymphoma cells, monoclonal antibodies (MAbs) were raised against variant S49 mouse lymphoma cells. Immunoperoxidase analysis with a specific MAb, named M-66, revealed nuclear localization with prominent staining in the nucleoli of both tumorigenic (T-63) cells and nontumorigenic, immunogenic (T-25-Adh) cells. Weak signals were also observed in the cytoplasm and plasma membrane of both cells. Western blot analysis with M-66 antibody revealed a 14-kDa protein in nuclear extracts of both T-25-Adh and T-63 cells. An additional nuclear 21-kDa protein was evident only in T-63 cells. M-66 identified several clones from a T-25-Adh cDNA expression library. These clones demonstrated extensive homology (approximately 95% identity throughout their length) to the mouse mammary tumor virus (MMTV) env and LTR regions. Extensive amino acid sequence homology (approximately 90% identity) between the clones and the env protein was observed. M-66 identified the 14-kDa protein in another MMTV bearing T-cell lymphoma, EL-4. Immunoperoxidase analysis of EL-4 cells with M-66 also revealed prominent nucleolar staining. MMTV-negative cells and MMTV-positive cells of nonlymphocytic origin were devoid of both 14- and 21-kDa proteins. Moreover, an anti-MMTV gp52 (env) antibody precipitated the 21-kDa protein in T-63 cells. We thus suggest that MMTV bearing T-cell lymphomas express nucleolar proteins translated from the env region of MMTV.
Amino Acid Sequence↗
Variant mouse lymphoma cells with modified response to interferon demonstrate enhanced immunogenicity.
We have previously developed an experimental model for the xenogenization of malignant lymphoma. From highly tumorigenic S49 mouse lymphoma cells that proliferate in suspension culture (designated T-25), we selected variant clones that grew as an adherent monolayer (designated T-25-Adh) and were non-tumorigenic in syngeneic mice. Furthermore, priming of syngeneic hosts with T-25-Adh cells protected them against subsequent challenges with the tumorigenic T-25 cells. Several lines of evidence have indicated that antigens of an endogenous mouse mammary tumor virus (MMTV) are involved in the immunogenicity of T-25-Adh cells. Since interferon (IFN) is known to affect retroviral assembly and maturation on the cell membrane, we have studied the effects of IFN on endogenous MMTV-related structures, as well as on the immunogenicity of T-25-Adh cells. We observed that mouse alpha and beta interferons affect the morphogenesis of intracellular MMTV-related precursors in the immunogenic T-25-Adh cells, but not in tumorigenic T-25 cells. From T-25-Adh cells we selected variants that were either high responders or low responders to the above-mentioned interferon effect. The high-response variants were significantly more protective against tumorigenic T-25 cells than the low-response variants. Involvement of MMTV-related antigens in the immune response of the host to T-25-Adh cells was further suggested by immunoelectron-microscopical analysis, demonstrating that antisera from mice, immunized with T-25-Adh cells, interacted specifically with cell-surface MMTV budding particles. These findings indicate a novel method for xenogenization of lymphoma cells by IFN. Since endogenous retroviruses are present in all tissues of the mouse, this approach might be applicable to a wide variety of tumors.
Animals↗
An experimental model for infiltration of malignant lymphoma to the eye and brain.
Currently there is no adequate experimental model available whereby the lethal infiltration of malignant lymphoma to the eye and CNS can be studied. Variant S49 mouse lymphoma cells that exhibit cell-cell adhesion properties (named Rev-2-T-6) were inoculated intraperitoneally into Balb/C mice at the ages of 6-60 days postnatal. Mice inoculated between days 6-11 postnatal developed signs of eye and CNS involvement with an apparent peak (58% of mice) at day 7. None of the mice inoculated beyond day 11 exhibited such signs. Histological analysis of these sites revealed tumorous infiltrates into a variety of structures in the orbit, intraocular tissues, along the optic nerve and in the brain. Additional analysis of the histopathological data, based on the structures demonstrating the highest frequency of lymphoma infiltration, suggests preferred routes of lymphoma entry to the brain and eye. Thus, entry to the brain can occur mainly through the choroid plexus and cranial nerves or cranial nerve ganglia. Entry to the eye may occur from the brain (along the optic nerve), and through hematogenous infiltration of orbital structures. No data were found that would support retrograde infiltration of the lymphoma from the eye to the brain. These findings present an experimental model for addressing the molecular mechanisms that govern homing of malignant lymphoma to the eye and brain, as well as the development of experimental therapeutic modalities for malignant lymphoma in these organs.
Animals↗
Debating dissociative diagnoses.
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Attitude of Health Personnel↗
End-tidal estimates of arterial PCO2 for cardiac output measurement by CO2 rebreathing: a study in patients with cystic fibrosis and healthy controls.
We set out to determine the effects of various estimates of arterial PCO2 (PaCO2) on calculation of cardiac output (Q) by the indirect Fick (CO2) method in healthy children and children with cystic fibrosis (CF), and to develop a prediction equation for children for PaCO2, based on end-tidal PCO2 (PetCO2). The study had 3 parts: 1) Twenty-three healthy children exercised lightly and moderately while arterialized capillary blood gases and PetCO2 were measured simultaneously so that a prediction equation for PaCO2 could be derived from PetCO2. Cardiac output was measured by CO2 rebreathing at each workload; different values for PaCO2 (measured in arterialized capillary blood, end-tidal, and PaCO2 derived from the Bohr equation assuming normal dead space) were used to calculate Q; 2) our equation PaCO2 = 0.647 PetCO2 + 12.4 was tested prospectively to measure Q in 9 healthy children; and 3) cardiac output based on arterialized capillary PaCO2 was compared with that based on Jones-corrected PetCO2 during light and moderate exercise in 16 CF patients whose forced expiratory volume in 1 second (FEV1), range from normal to 37% predicted. Our results have shown that in healthy children end-tidal based-estimates of PaCO2 tended to overestimate Q, whereas PaCO2 values derived by the Bohr equation and assuming normal dead space tended to underestimate Q, compared with Q calculated from directly measured PaCO2. Our prediction equation resulted in good agreement compared with directly measured PaCO2 when used to calculate Q (mean difference, +1.3%; range, +9% to -13%). CF patients with little or no airway obstruction had results similar to healthy controls, but those with severe airway obstruction had lower values for Q when PetCO2 was used instead of directly measured PaCO2. We conclude that estimates of PaCO2 from PetCO2 are not reliable in patients with moderately severe pulmonary disease due to CF. In healthy children, the prediction equation for PaCO2 from PetCO2 derived in the present study gives results superior to other bloodless methods currently in use for computation of Q by the indirect Fick (CO2) method.
Adolescent↗
KT-5720 reverses multidrug resistance in variant S49 mouse lymphoma cells transduced with the human MDR1 cDNA and in human multidrug-resistant carcinoma cells.
T-25-Adh cells, cell variants derived from S49 mouse lymphoma, were transduced with a retrovirus containing the human MDR1 cDNA. The resultant cells (HU-1) are cross-resistant to colchicine, doxorubicin, vinblastine and actinomycin D, and their resistance to colchicine is reversed by verapamil. HU-1 cells were used to screen several protein kinase modulators for their ability to reverse multidrug resistance. Among the tested indole carbazole (K-252a) family of protein kinase inhibitors, only the antibiotic alkaloid KT-5720 (9-n-hexyl derivative of K-252a) could overcome the multidrug resistance of HU-1 cells and KB-V1 human carcinoma cells. Since other protein kinase A, C and G modulators did not reverse multidrug resistance in the tested multidrug-resistant cells, the chemosensitising activity of KT-5720 on these cells is apparently independent of its kinase inhibitory effects. Since KT-5720 fully reversed multidrug resistance at non-toxic concentrations, it might be a candidate for clinical chemosensitisation in combination chemotherapy.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Association of tumorigenic and nontumorigenic (immunogenic) variants in a mouse T-cell lymphoma with two distinct p53 mutations.
An in vitro model system for xenogenization has been developed in which an immunogenic, nonmalignant phenotype was selected from a highly malignant T-cell line (S49). We showed by single-strand conformation polymorphism and DNA sequence analysis that specific point mutations in the p53 tumor suppressor gene correlated with a change from a tumorigenic to a nontumorigenic (immunogenic) phenotype. Specifically, we found that the highly malignant S49 cell line T-60 contains an Arg-->Gln substitution at residue 246 in exon 7 of p53. In contrast, nontumorigenic (immunogenic) variants (T-25-Adh and Rev-1) exhibited a Gly-->Ser substitution at residue 242 of p53. In two subsequent tumorigenic revertants derived from Rev-1, we again found the Arg-->Gln substitution at residue 246 that was found initially in the T-60 cells. Thus, mutation at residue 246 of p53 was associated with a highly malignant phenotype, whereas a novel mutation at residue 242 of p53 appeared to be associated with a nonmalignant phenotype and may have actually protected the host through immunization. We conclude that mutation of residue 242 may represent a new class of permissive (nonmalignant) mutations in the mouse that are analogous to the Li-Fraumeni mutation in humans.
Amino Acid Sequence↗
Conditionally immortalized intestinal epithelial cells: novel approach for study of differentiated enterocytes.
Clonal cell lines have been established from primary fetal rat intestinal epithelial cells by stable transfection with plasmids containing either the simian virus 40 (SV40) large T-antigen gene under the control of the heavy metal inducible metallothionein promoter (pMTWt) or the thermolabile SV40 T-antigen gene under the control of the SV40 early promoter (pZipSVtsa58). pMTWt-transfected cells produced sufficient T-antigen to allow them to proliferate both when the metallothionein promoter was induced and uninduced. No differences were observed in the pattern of intestinal epithelial markers expressed when the cells were cultured in the presence or absence of inducing agent (zinc). In contrast, fetal rat intestinal epithelial cells transfected with pZipSVtsa58 were immortalized conditionally; cells proliferated at 32 degrees C but ceased to proliferate between 48 and 72 h of culture at 39 degrees C. Four of these cell lines were characterized in detail; they showed microvilli and tight junctions as well as dome formation and expressed functional and biochemical markers of intestinal epithelial cells, including keratins 8, 19, and 21, aminopeptidase N, and dipeptidyl peptidase IV. One cell line, 2/4/A1, expressed in addition a low level of lactase and sucrase-isomaltase. The amount and/or activity of some of these markers changed during the switch from the proliferative to the nonproliferative state (switch from culture at 32 to 39 degrees C), resulting in a more differentiated phenotype and mimicking similar changes taking place during intestinal epithelial cell differentiation in vivo.
Animals↗
Monoclonal antibodies to immunogenic lymphoma cell variants displaying impaired neoplastic properties: characterization and applications.
Immunogenic, nontumorigenic cell variants derived from the highly tumorigenic mouse lymphoma cell line S-49 were used to raise monoclonal antibodies (MAbs) in syngeneic BALB/c mice. MAbs of the following specifications were derived: (a) MAbs that interacted preferentially with the immunogenic variants, (b) MAbs that interacted with both immunogenic variants and parental tumorigenic cells, and (c) a MAb that interacted with both immunogenic and tumorigenic S-49 cells and the normal BALB/c splenocytes. Six MAbs raised in this way were found to recognize at least five different cell-surface epitopes. Functional analysis of the different MAbs suggested their potential usefulness in passive immunization against parental tumorigenic cells as well as in enrichment of immunogenic cells from a mixed population containing a preponderance of tumorigenic cells.
Animals↗
Synthesis, location, and lateral mobility of fluorescently labeled ubiquinone 10 in mitochondrial and artificial membranes.
To explore the influence of the long isoprene chain of ubiquinone 10 (UQ) on the mobility of the molecule in a phospholipid bilayer, we have synthesized a fluorescent derivative of the head-group moiety of UQ and measured its lateral diffusion in inner membranes of giant mitochondria and in large unilamellar vesicles. The diffusion coefficients, determined by the technique of fluorescence redistribution after photobleaching, were 3.1 X 10(-9) cm2 s-1 in mitochondria and 1.1 X 10(-8) cm2 s-1 in vesicles. Similar diffusion rates were observed for fluorescently labeled phosphatidylethanolamine (PE) with the same moiety attached to its head group (4-nitro-2,1,3-benzooxadiazole: NBD). Fluorescence emission studies carried out in organic solvents of different dielectric constants, and in vesicles and mitochondrial membranes, indicate that NBDUQ is located in a more hydrophobic environment than NBDPE or the starting material IANBD (4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitro-2,1,3- benzoxadiazole). Fluorescence quenching studies carried out with CuSO4, a water-soluble quenching agent, also indicate that NBDUQ is located deeper in the membrane than NBDPE. These results suggest that ubiquinone and PE are oriented differently in a membrane, even though their diffusion rates are similar. Conclusions regarding whether or not diffusion of UQ is a rate-limiting step in electron transfer must await a more detailed knowledge of the structural organization and properties of the electron transfer components.
4-Chloro-7-nitrobenzofurazan↗
Immunohistochemical analysis of the segregation process of the quail germ cell lineage.
An antiserum against quail 7 day gonadal germ cells was found to react specifically with gonadal germ cells of both sexes. Transverse sections from a range of early quail developmental stages were submitted to the antibody PAP reaction. Blastodiscs from the earliest uterine stages (II to X E.G. & K) reacted very strongly, while the overall reaction gradually decreased in older blastoderms. At stage XIII both epiblast and hypoblast were weakly stained, but some large, PGC-like cells stained intensively. During gastrulation (PS formation) the reaction of the epiblast disappears quicker than that of the hypoblast. The newly formed mesoderm and entoderm do not react at all and the reaction gradually becomes limited mainly to the PGCs and somewhat to the primary hypoblast which is moving into the germinal crescent. The widely spread reaction at the early stages is thus gradually being restricted to the PGCs.
Animals↗
Isolation and characterization of interferon-resistant variants from S49 mouse lymphoma.
S49 mouse lymphoma cells were found to be extremely sensitive to the antiproliferative activity of interferon. These characteristics were studied to select for IFN-resistant cell variants. Some 0.6% of the parental S49 cell population were resistant to the antiproliferative and cytotoxic activities of IFN. The resistant cells were cloned and analyzed for their responses to several of the activities of IFN, namely, inhibition of encephalomyocarditis (EMC) virus, murine leukemia virus (MuLV) replications, and the induction of (2'-5') oligoadenylate synthetase. Among the clones selected some were highly resistant while others demonstrated only partial responsiveness to IFN. S49 cells demonstrate tubular structures in the cytoplasm. These structures were previously reported to be antigenically related to mouse mammary tumor virus (MMTV). We report here that IFN treatment decreases the expression of these cytoplasmic viral structures as revealed by electron microscopy. To correlate this novel antiviral activity to the more established functions of IFN we utilized the above mentioned S49 IFN-resistant variants. The anti-MMTV activity of IFN correlated with the other effects of IFN in both the highly resistant and partially responsive S49 clones. Our findings indicate that a relatively high proportion of S49 cells vary in their response to IFN. The defect in the resistant cells appears to affect a primary response to IFN which is common to its diverse activities. Furthermore, the effect of IFN on MMTV-related structures involves the usual pathway of IFN action.
2',5'-Oligoadenylate Synthetase↗