Taxol increases steady-state levels of lipopolysaccharide-inducible genes and protein-tyrosine phosphorylation in murine macrophages.

Taxol, a microtubule stabilizing agent, exhibits promise in the treatment of breast and ovarian tumors. Recently, this novel drug has been shown to activate murine macrophages to express TNF-alpha and to down-regulate TNF-alpha receptors, activities shared by bacterial LPS. Our study sought to determine if taxol could regulate gene expression in murine macrophages and to examine further the ability of taxol to generate an LPS-like signal. Toward this end, the ability of taxol to induce TNF-alpha mRNA and five other genes (IL-1 beta, IP-10, D3, D7, and D8) associated with LPS-activation of macrophages was examined by Northern blot analysis. Taxol alone (1-30 microM) induced murine C3H/OuJ macrophages to secrete bioactive TNF-alpha and express increased levels of each of the six genes under investigation. The magnitude and the kinetics of induction of each gene closely resembled that seen with Escherichia coli K235 LPS. Macrophages from LPS-hyporesponsive C3H/HeJ mice, however, failed to induce detectably any of the genes in response to taxol, despite being sensitive to the microtubule stabilizing effects of taxol as determined by immunofluorescence microscopy. The gene induction activity of taxol was in marked contrast to an alternative macrophage activator, heat killed Staphylococcus aureus, which induced a distinct gene profile in C3H/OuJ macrophages and which was equally active in C3H/OuJ and C3H/HeJ macrophages. These data are consistent with an ability of taxol to generate an LPS-like signal, possibly through a common signaling intermediate. As a first step toward identifying signal responses shared by taxol and LPS, we have shown that taxol, as shown previously for LPS, rapidly induces the tyrosine phosphorylation of a 41- and 42-kDa protein.     

A TEA-insensitive flickering potassium channel active around the resting potential in myelinated nerve

A novel potassium-selective channel which is active at membrane potentials between -100 mV and +40 mV has been identified in peripheral myelinated axons of Xenopus laevis using the patch-clamp technique. At negative potentials with 105 mM-K on both sides of the membrane, the channel at 1 kHz resolution showed a series of brief openings and closings interrupted by longer closings, resulting in a flickery bursting activity. Measurements with resolution up to 10 kHz revealed a single-channel conductance of 49 pS with 105 mM-K and 17 pS with 2.5 mM-K on the outer side of the membrane. The channel was selective for K ions over Na ions (PNa/PK = 0.033). The probability of being within a burst in outside-out patches varied from patch to patch (> 0.2, but often > 0.9), and was independent of membrane potential. Open-time histograms were satisfactorily described with a single exponential (tau o = 0.09 msec), closed times with the sum of three exponentials (tau c = 0.13, 5.9, and 36.6 msec). Sensitivity to external tetraethylammonium was comparatively low (IC50 = 19.0 mM). External Cs ions reduced the apparent unitary conductance for inward currents at Em = -90 mV (IC50 = 1.1 mM). Ba and, more potently, Zn ions lowered not only the apparent single-channel conductance but also open probability. The local anesthetic bupivacaine with high potency reduced probability of being within a burst (IC50 = 165 nM). The flickering K channel is clearly different from the other five types of K channels identified so far in the same preparation. We suggest that this channel may form the molecular basis of the resting potential in vertebrate myelinated axons.     

A mutation in the pro alpha 2(I) gene (COL1A2) for type I procollagen in Ehlers-Danlos syndrome type VII: evidence suggesting that skipping of exon 6 in RNA splicing may be a common cause of the phenotype.

Fibroblasts from a proband with Ehlers-Danlos syndrome type VII synthesized approximately equal amounts of normal and shortened pro alpha 2(I) chains of type I procollagen. Nuclease S1 probe protection experiments with mRNA demonstrated that the pro alpha 2(I) chains were shortened because of a deletion of most or all of the 54 nucleotides in exon 6, the exon that contains codons for the cleavage site for procollagen N-proteinase. Sequencing of genomic clones revealed a single-base mutation that converted the first nucleotide of intron 6 from G to A. Therefore, the mutation was a change, in the -GT-consensus splice site, that produced efficient exon skipping. Allele-specific oligonucleotide hybridizations demonstrated that the proband's mother, father, and brother did not have the mutation. Therefore, the mutation was a sporadic one. Analysis of potential 5' splice sites in the 5' end of intron 6 indicated that none had favorable values by the two commonly employed techniques for evaluating such sites. The proband is the fourth reported proband with Ehlers-Danlos syndrome VII with a single-base mutation that causes skipping of exon 6 in the splicing of RNA from either the COL1A1 gene or COL1A2 gene. No other mutations in the two type I procollagen genes have been found in the syndrome. Therefore, such mutations may be a common cause of the phenotype. The primers developed should be useful in screening for the same or similar mutations causing the disease.     

Kinetics of aerobic biodegradation of benzene and toluene in sandy aquifer material

Monod's equation adequately described aerobic biodegradation rates of benzene and toluene by the microbial population of a sandy aquifer when these compounds were initially present at concentrations lower than 100 mg/l each. Concentrations higher than 100 mg/l were inhibitory, and no benzene or toluene degradation was observed when these compounds were initially present at 250 mg/l each. The Monod coefficients were calculated as k = 8.3 g-benzene/g-cells/day and Ks = 12.2 mg/l for benzene, and k = 9.9 g-toluene/g-cells/day and Ks = 17.4 mg/l for toluene. Specific first-order coefficients would be 0.68 l/mg.day for benzene and 0.57 l.mg.day for toluene.     

Phenotypic switching in cells transformed with the herpes simplex virus thymidine kinase gene

Biochemical transformation of Ltk- cells with the herpes simplex virus thymidine kinase (tk) gene resulted in numerous TK+ colonies that survived selection in hypoxanthine-aminopterin-thymidine medium. Many of these TK+ cell lines switched phenotypes and reverted to the TK- state. In this report, we describe the biological and biochemical characteristics of three TK- revertant lines. One (K1B5) transiently expressed TK in the presence of bromodeoxyuridine, which selects for the TK- phenotype. Another TK- sibling (K1B6n) expressed TK only after removal from bromodeoxyuridine-containing medium. The last variant (K1B6me) lost the ability to switch to the TK+ phenotype, although it maintained the herpes simplex virus sequences coding for TK. Loss of the ability of K1B6me cells to express TK was correlated with extensive methylation of the sequence recognized by the restriction endonuclease HpaII (pCpCpGpG). After these cells were treated with 5-azacytidine, they regained the ability to clone in hypoxanthine-aminopterin-thymidine medium and reexpressed virus tk mRNA and enzyme. In addition, the HpaII sites that were previously shown to be refractile to enzyme digestion were converted to a sensitive state, demonstrating that they were no longer methylated.     

Position of chromosomes in the human interphase nucleus

Summary The problem of localization of chromosomes in relation to each other in the interphase nucleus of human lymphocytes was investigated by analysis of chromatid and chromosome aberrations observed in lymphocyte cultures of three patients with Fanconi's anemia, one patient with Bloom's syndrome, and in Trenimon-treated (Trenimon, Bayer) normal cells. Distribution of open gaps and breaks is highly correlated with chromosome length and distribution of breaks involved in chromatid translocations in Fanconi's anemia and in Trenimontreated cells. Both correlations are much lower in Bloom's syndrome. In Fanconi's anemia and in normal cells after Trenimon-treatment, the majority of chromatid translocations are between nonhomologous chromosomes, whereas in Bloom's syndrome mainly homologous chromosomes are involved. Statistical localization of chromosomes in relation to each other in the three-dimensional space by multidimensional scaling gives results consistent with the limited amount of independent evidence.     

Defective Fc-mediated phagocytosis in C3H/HeJ macrophages. II. Correction by cAMP agonists

Peritoneal macrophages from LPS hyporesponsive C3H/HeJ mice lose the capacity to bind and phagocytose opsonized sheep erythrocytes (EA) over a 48-hr culture period. This loss in Fc receptor capacity is markedly different from the progressive increase in phagocytic ability exhibited by cultured macrophages derived from LPS-responsive C3H/HeN mice. Since dibutyryl-cyclic adenosine monophosphate (DBcAMP) has previously been reported to modulate membrane receptor expression in lymphocytes and certain macrophage-like cell lines, we examined its effects on EA binding and phagocytosis by C3H/HeJ macrophages. DBcAMP not only reverses the binding defect in C3H/HeJ macrophages but also restores EA phagocytosis to the level of control C3H/HeN cultures. 8-Bromo-cAMP, as well as other agents known to elevate intracellular cAMP (i.e., isoproterenol plus isobutylmethylxanthine or prostaglandin E2) also corrected the phagocytic defect. Since the C3H/HeJ macrophage phagocytic defect can also be reversed by in vitro stimulation with a lymphokine-rich culture supernatant, we examined the effect of this treatment on intracellular cAMP levels. Lymphokine treatment produced a 60% increase in the levels of macrophage intracellular cAMP. These findings suggest that the C3H/HeJ differentiation defect may be secondary to some abnormality in a cAMP dependent pathway.     

EEG differences in neurotic as compared with normal twin pairs

Summary The resting EEGs of 17 twin pairs originally traced through one neurotic co-twin (10 monozygotic and 7 dizygotic pairs aged between 18 and 63 years) have been described and compared with the neuroticism scores (Schepank, 1974) of these twins. EEG comparison according to the customary visual criteria failed to show any consistent EEG differences between monozygotic co-twins, whereas dizygotic pairs often showed EEG discordance. Computerized time-domain (interval-amplitude) analysis failed to show a higher degree of EEG discordance between neurotic MZ cotwins than between co-twins in 25 adult nonneurotic male MZ pairs (age range 18–33; mean age 22.9 years). There were no significant correlations between EEG differences and differences in the neuroticism score among ten MZ pairs traced through a neurotic co-twin. It is concluded that the individual and genetically determined EEG pattern is manifest even in the face of the long-lasting psychological alterations observed in neurotics.