Interleukin-4 downregulates the p70 chain of the IL-2 receptor on peripheral blood mononuclear cells

Data that support a differential regulation of the interleukin-2 receptor (IL-2R) alpha-(p55 or Tac) and beta-chain (p70) expression by IL-4 are presented. Cytofluorometric analysis performed on peripheral blood mononuclear cells, some of which had been nylon wool passed (enriched for T-cells), in the presence or absence of phytohemagglutinin (PHA) or OKT3, demonstrated that IL-4 has a dose-dependent capacity to inhibit beta-chain IL-2R expression, whereas the alpha-chain is nearly unaffected. We could also, as a consequence of the decreased p70 expression, detect a slight increase in the amounts of IL-2 obtained from PHA-stimulated cultures, when IL-4 was present. Further, the proliferative response, especially to IL-2, but also to PHA alone, was depressed in the presence of IL-4. These data thus give further support to the idea that not only the IL-2R complex as such, but also the two individual IL-2R chains, can be independently regulated.     

The preparation of biotinyl-epsilon-aminocaproylated forms of the vasoactive intestinal polypeptide (VIP) as probes for the VIP receptor.

Vasoactive intestinal polypeptide (VIP) was biotinyl-epsilon-aminocaproylated using sulfosuccinimidyl-6-(biotinamido) hexanoate thereby producing a series of products that were separated by high performance liquid chromatography (HPLC). Seven VIP-derivatives were isolated and the number and location of biotinyl-epsilon-aminocaproylation was determined by a combination of enzymatic degradation and plasma desorption mass spectrometry (PDMS). Receptor binding experiments with the VIP biotinyl-epsilon-aminocaproylated derivatives revealed IC50 values for the monobiotinyl-epsilon-aminocaproylated peptides that were 1.3-3.2 times higher than for natural VIP. All isolated biotinyl-epsilon-aminocaproylated derivatives possess VIP-like bioactivity as shown by an assay measuring pancreatic juice secretion in cat, VIP biotinyl-epsilon-aminocaproylated in position lysine being almost equipotent with natural VIP.     

Methane monooxygenase component B and reductase alter the regioselectivity of the hydroxylase component-catalyzed reactions. A novel role for protein-protein interactions in an oxygenase mechanism.

The soluble methane monooxygenase (MMO) system, consisting of reductase, component B, and hydroxylase (MMOH), catalyzes NADH and O2-dependent monooxygenation of many hydrocarbons. MMOH contains 2 mu-(H or R)oxo-bridged dinuclear iron clusters thought to be the sites of catalysis. Although rapid NADH-coupled turnover requires all three protein components, three less complex systems are also functional: System I, NADH, O2, reductase, and MMOH; System II, H2O2 and oxidized MMOH; System III, MMOH reduced nonenzymatically by 2e- and then exposed to O2 (single turnover). All three systems give the same products, suggesting a common reactive oxygen species. However, the distribution of products observed for most substrates that are hydroxylated in more than one position is different for each system. For several of these substrates, addition of component B to Systems I, II, or III causes the product distributions to shift dramatically. These shifts result in identical product distributions for Systems I and III in which MMOH passes through the 2e- reduced state ([Fe(II).Fe(II)]) during catalysis. In contrast, System II (in which MMOH probably does not become reduced) generally gives a unique product distribution. It is proposed that changes in MMOH structure occurring upon diiron cluster reduction and/or component complex formation cause substrates to be presented differently to the activated oxygen species. Kinetic studies show that component B strongly activates System I and, in most cases, strongly deactivates System II. The effect of component B on product distribution of System I (and III) occurs at less than 5% of the MMOH concentration, while nearly stoichiometric concentrations are required to maximize the rate of System I. This shows that component B has at least two roles in catalysis. EPR monitored titration of reduced MMOH ([Fe(II).Fe(II)]) with component B suggests that the effect of substoichiometric component B on product distribution is due to hysteresis in the MMOH conformational changes.     

Induction of chromosomal aberrations by camptothecin in root-tip cells of Vicia faba.

When root-tip cells of Vicia faba were exposed during early and middle interphase to camptothecin (Cpt), the aberrations obtained were exclusively of the chromatid type and tended to be localized in late replicating heterochromatic regions of the chromosomes. In these respects the clastogenic effect of Cpt resembles that of agents that act by an S-phase-dependent mechanism. In contrast to typical S-phase-dependent agents, Cpt produced lesions capable of giving rise to aberrations only in S-phase cells that were synthesizing DNA at the time of treatment. The dependence on ongoing DNA synthesis was suggested in autoradiographic experiments and by the fact that the clastogenic effect of Cpt was strongly suppressed by hydroxyurea, an inhibitor of DNA synthesis. After Cpt treatments, there were many more cells with 3-12 aberrations and far fewer cells with 0, 1 or 2 aberrations than expected on the basis of a Poisson distribution. Cpt further differed from typical S-phase-dependent agents by being capable of inducing lesions in the G2 phase that give rise to chromosomal aberrations in the first mitosis after treatment. This effect was obtained at Cpt concentrations around 10 microM, whereas only 0.03 microM Cpt was required to produce chromatid aberrations in the S phase. Results of G2-phase experiments with Cpt and 5-fluorodeoxyuridine, an inhibitor of DNA synthesis, suggest that DNA synthesis is required for the clastogenic effect of Cpt not only during the S phase, but also during the G2 phase, although the DNA syntheses involved are both quantitatively and qualitatively different.     

Structure-activity relationships for unsaturated dialdehydes. 6. The mutagenic activity of 11 compounds in the V79/HGPRT assay.

The mutagenic activity of 11 sesquiterpenoid unsaturated dialdehydes in the V79/HGPRT assay has been determined, and is compared with previously published data on the mutagenicity of the same compounds towards Ames Salmonella strains. One compound, isovelleral, is a potent mutagen in both assays, while six compounds, which are positive in the Ames Salmonella/microsome assay, show no significant activity in this study. One compound, acetylmerulidial, is negative in the Ames Salmonella/microsome assay but significantly although weakly mutagenic in the V79/HGPRT assay. The remaining three compounds are inactive in both assays. The study is part of a general investigation of quantitative structure-activity relationships for unsaturated dialdehydes, a class of natural occurring compounds known for their potent and numerous biological activities.     

Screening for mutations in human HPRT cDNA using the polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis (CDGE).

Previously, we reported the modification of denaturing gradient gel electrophoresis called constant denaturant gel electrophoresis (CDGE). CDGE separates mutant fragments in specific melting domains. CDGE seems to be a useful tool in mutation detection. Since the hypoxanthine phosphoribosyltransferase (HPRT) gene is widely used as target locus for mutation studies in vitro and in vivo, we have examined the approach of analyzing human HPRT cDNA by polymerase chain reaction (PCR) and CDGE. All nine HPRT exons are included in a 716-bp cDNA fragment obtained by PCR using HPRT cDNA as template. When the full-length cDNA fragment was examined by CDGE, it was possible to detect mutations only in the last part of exon 8 and exon 9. However, digestion of the cDNA fragment with the restriction enzyme AvaI prior to CDGE enabled us to detect point mutations in most of exon 2, the beginning of exon 3, the last part of exon 8 and exon 9. With the use of two internal primer sets, including a GC-rich clamp on one of the primers in each pair, a region containing most of exon 3 through exon 6 was amplified and we were able to resolve fragments with point mutations in this region from wild-type DNA. The approach described here allows for rapid screening of point mutations in about two thirds of the human HPRT cDNA sequence. In a test of this approach, we were able to resolve 12 of 13 known mutants. The mutant panel included one single-base deletion, one two-base deletion and 11 single-base substitutions.     

Floral display and pollination success in Achillea ptarmica (Asteraceae)

In a natural population of the self-incompatible Achillea ptarmica , many-headed inflorescences attracted most pollinators (flies), and each pollinator visited more heads on large inflorescences than on smaller ones. The visitation rate did not increase disproportionately with inflorescence size, as would be expected if clusters of heads had a synergetic effect on pollinator attraction. Both the calculated visitation rate per head and the percentage seed set increased slightly as a function of head number. The removal of rays from all heads in the inflorescence reduced the approach rate by 51%, but had only a small negative effect on seed set (12%). Hence, ray removal probably affected male fertility (pollen donation) to a greater extent than female fertility. The decline in pollination success was similar for all inflorescence sizes, suggesting that ray and inflorescence size have independent effects on the total display, and thus represent separate targets for pollinator-mediated selection. Residual variation in seed set was strongly correlated with patch identity, indicating clonal or environmentally induced differences in female reproductive success.     

Restoration of light induced photosystem II inhibition without de novo protein synthesis

Illumination of isolated spinach thylakoid membranes under anaerobic conditions gave rise to severe inhibition of photosystem II electron transport but did not result in D1-protein degradation. When these photoinhibited thylakoids were incubated in total darkness the photosystem II activity could be fully restored in vitro in a process that required 1-2 h for completion.     

Cloning, structure, and expression of a rat binding protein for polychlorinated biphenyls. Homology to the hormonally regulated progesterone-binding protein uteroglobin

Certain metabolites of polychlorinated biphenyls (PCBs) are retained in the Clara cells and in the airway lumen of rodent lung due to their interaction with a secretory 13-kDa protein. Here, we report the isolation of a cDNA encoding the rat lung PCB-binding protein. The identity of the PCB-binding protein is supported by expression of the cDNA in Cos-1 cells where the homogenates from transfected cells show specific binding of 4,4'-bis([ 3H]methylsulfonyl)-2,2',5,5'-tetrachlorobiphenyl, a high affinity ligand for the PCB-binding protein. Also a monospecific antiserum to the PCB-binding protein recognizes a 13-kDa protein in the homogenates of transfected cells but not in the corresponding fraction of mock-transfected cells. Northern blot analysis of total RNA from different rat tissues demonstrates that the cDNA detects a approximately 600-base pair mRNA which appears to be solely expressed in lung. Interestingly, DNA sequence analysis and prediction of the amino acid sequence reveals that the PCB-binding protein shares 53% positional amino acid identity with uteroglobin, a progesterone-binding protein found in rabbit uterus and lung. Furthermore, amino acids shown by x-ray crystallography to delineate the central cavity of uteroglobin, which fits progesterone, are highly conserved in the two proteins.     

Similar induction of the hepatic EGF receptor in vivo by EGF and partial hepatectomy

A 2-fold increase in the level of epidermal growth factor (EGF) receptor mRNA accompanied by a similar increase in newly synthesized ligand-binding EGF-receptors was observed 2-4 h after intraportal EGF-injections and 2-4 h after partial hepatectomy. After this initial increase, the EGF-receptor levels decreased back to control levels 6-8 h after EGF-injections and below control levels 6-8 h after partial hepatectomy. EGF was also found to influence the degradation of endocytosed [125I]-EGF 3 h after injection to a similar extent as partial hepatectomy. The similar effects of EGF and partial hepatectomy suggests that EGF or EGF-like factors may be mechanistically involved in the early "promotion" stage during the pre-replicative phase of liver regeneration. The EGF-induced effects are, however, at least not alone responsible for the replicative process.