Eosinophil degranulation. An immunologic determinant in the pathogenesis of the Mazzotti reaction in human onchocerciasis

Onchocerciasis patients treated with diethylcarbamazine often undergo a severe inflammatory response, the Mazzotti reaction. To assess the eosinophil's role in the pathogenesis of the Mazzotti reaction, we obtained serial blood, plasma, and skin biopsy specimens from 21 heavily infected patients and 3 endemic controls, both before and during therapy with diethylcarbamazine. Samples were analyzed for blood eosinophils, plasma levels of eosinophil granule major basic protein (MBP) and eosinophil-derived neurotoxin, eosinophil infiltration and eosinophil and mast cell degranulation in the skin. After the first dose of diethylcarbamazine, blood eosinophils fell from a pre-treatment level of 888 +/- 111 to 203 +/- 42 cells/mm3 at 8 h. This decrease was followed by a marked eosinophilia developing over the remaining 7 days of treatment and 14 days of follow-up. Plasma eosinophil-derived neurotoxin levels increased from 56 +/- 4 ng/ml pretreatment to a peak of 82 +/- 9 ng/ml at 8 h and returned to pretreatment levels by 48 h. Beginning at 12 h, plasma MBP levels increased from 730 +/- 74 ng/ml pretreatment to a peak of 1140 +/- 74 ng/ml after 5 days. Pretreatment skin biopsies stained for MBP by immunofluorescence showed a bright fibrillar pattern in the dermis consistent with chronic eosinophil degranulation; the MBP was localized on elastic tissue fibers. After treatment, skin biopsy specimens showed both the pretreatment fibrillar MBP staining pattern as well as focal eosinophil degranulation. Deposition of MBP around microfilariae in the papillary dermis was visible as early as 1.5 h. The lowest blood eosinophil levels and peak plasma eosinophil-derived neurotoxin levels coincided with the infiltration and degranulation of eosinophils in the skin. Mast cell degranulation in the skin was maximal by the first posttreatment biopsy (1.5 h) coincident with the beginning of eosinophil degranulation. Although the pathogenesis of the Mazzotti reaction is clearly complex, our results indicate that eosinophil degranulation is characteristic of the response and that it occurs with a time course suggestive of a role for the eosinophil in determining the clinical and pathologic manifestations of the reaction.     

Protein phosphatase 6 regulates mitotic spindle formation by controlling the T-loop phosphorylation state of Aurora A bound to its activator TPX2

Many protein kinases are activated by a conserved regulatory step involving T-loop phosphorylation. Although there is considerable focus on kinase activator proteins, the importance of specific T-loop phosphatases reversing kinase activation has been underappreciated. We find that the protein phosphatase 6 (PP6) holoenzyme is the major T-loop phosphatase for Aurora A, an essential mitotic kinase. Loss of PP6 function by depletion of catalytic or regulatory subunits interferes with spindle formation and chromosome alignment because of increased Aurora A activity. Aurora A T-loop phosphorylation and the stability of the Aurora A-TPX2 complex are increased in cells depleted of PP6 but not other phosphatases. Furthermore, purified PP6 acts as a T-loop phosphatase for Aurora A-TPX2 complexes in vitro, whereas catalytically inactive mutants cannot dephosphorylate Aurora A or rescue the PPP6C depletion phenotype. These results demonstrate a hitherto unappreciated role for PP6 as the T-loop phosphatase regulating Aurora A activity during spindle formation and suggest the general importance of this form of regulation.     

Phosphodiesterase-5 Gln817 is critical for cGMP, vardenafil, or sildenafil affinity: its orientation impacts cGMP but not cAMP affinity

The side group of an invariant Gln in cGMP- and cAMP-specific phosphodiesterases (PDE) is held in different orientations by bonds with other amino acids and purportedly discriminates between guanine and adenine in cGMP and cAMP. In cGMP-specific PDE5, Gln(775) constrains the orientation of the invariant Gln(817) side chain, which forms bidentate bonds with 5'-GMP, vardenafil, sildenafil, and 3-isobutyl-1-methylxanthine (IBMX) (Sung, B. J., Hwang, K. Y., Jeon, Y. H., Lee, J. I., Heo, Y. S., Kim, J. H., Moon, J., Yoon, J. M., Hyun, Y. L., Kim, E., Eum, S. J., Park, S. Y., Lee, J. O., Lee, T. G., Ro, S., and Cho, J. M. (2003) Nature 425, 98-102; Huai, Q., Liu, Y., Francis, S. H., Corbin, J. D., and Ke, H. (2004) J. Biol. Chem. 279, 13095-13101; Zhang, K. Y., Card, G. L., Suzuki, Y., Artis, D. R., Fong, D., Gillette, S., Hsieh, D., Neiman, J., West, B. L., Zhang, C., Milburn, M. V., Kim, S. H., Schlessinger, J., and Bollag, G. (2004) Mol. Cell 15, 279-286). PDE5(Q817A) and PDE5(Q775A) were generated to test the hypotheses that Gln(817) is critical for cyclic nucleotide or inhibitor affinity and that Gln(775) immobilizes the Gln(817) side chain to provide cGMP/cAMP selectivity. Allosteric cGMP binding and the molecular mass of the mutant proteins were unchanged compared with PDE5(WT). For PDE5(Q817A), K(m) for cGMP or cAMP was weakened 60- or 2-fold, respectively. For PDE5(Q775A), K(m) for cGMP was weakened approximately 20-fold but was unchanged for cAMP. For PDE5(Q817A), vardenafil, sildenafil, and IBMX inhibitory potencies were weakened 610-, 48-, and 60-fold, respectively, indicating that Gln(817) is a major determinant of potency, especially for vardenafil, and that binding of vardenafil and sildenafil differs substantially. Sildenafil and vardenafil affinity were not significantly affected in PDE5(Q775A). It is concluded that Gln(817) is a positive determinant for PDE5 affinity for cGMP and several inhibitors; Gln(775), which perhaps restricts rotation of Gln(817) side chain, is critical for cGMP affinity but has no measurable effect on affinity for cAMP, sildenafil, or vardenafil.     

Genetic Resistance to Scrapie Infection in Experimentally Challenged Goats

In goats, several field studies have identified coding mutations of the gene encoding the prion protein (I/M₁₄₂, N/D₁₄₆, S/D₁₄₆, R/Q₂₁₁, and Q/K₂₂₂) that are associated with a lower risk of developing classical scrapie. However, the data related to the levels of resistance to transmissible spongiform encephalopathies (TSEs) of these different PRNP gene mutations are still considered insufficient for developing large-scale genetic selection against scrapie in this species. In this study, we inoculated wild-type (WT) PRNP (I₁₄₂R₁₅₄R₂₁₁Q₂₂₂) goats and homozygous and/or heterozygous I/M₁₄₂, R/H₁₅₄, R/Q₂₁₁, and Q/K₂₂₂ goats with a goat natural scrapie isolate by either the oral or the intracerebral (i.c.) route. Our results indicate that the I/M₁₄₂ PRNP polymorphism does not provide substantial resistance to scrapie infection following intracerebral or oral inoculation. They also demonstrate that H₁₅₄, Q₂₁₁, and K₂₂₂ PRNP allele carriers are all resistant to scrapie infection following oral exposure. However, in comparison to WT animals, the H₁₅₄ and Q₂₁₁ allele carriers displayed only moderate increases in the incubation period following i.c. challenge. After i.c. challenge, heterozygous K₂₂₂ and a small proportion of homozygous K₂₂₂ goats also developed the disease, but with incubation periods that were 4 to 5 times longer than those in WT animals. These results support the contention that the K₂₂₂ goat prion protein variant provides a strong but not absolutely protective effect against classical scrapie.     

Proton-dependent electron transfer from CuA to heme a and altered EPR spectra in mutants close to heme a of cytochrome oxidase

Eukaryotic cytochrome c oxidase (CcO) and homologous prokaryotic forms of Rhodobacter and Paraccocus differ in the EPR spectrum of heme a. It was noted that a histidine ligand of heme a (H102) is hydrogen bonded to serine in Rhodobacter (S44) and Paraccocus CcOs, in contrast to glycine in the bovine enzyme. Mutation of S44 to glycine shifts the heme a EPR signal from g(z) = 2.82 to 2.86, closer to bovine heme a at 3.03, without modifying other properties. Mutation to aspartate, however, results in an oppositely shifted and split heme a EPR signal of g(z) = 2.72/2.78, accompanied by lower activity and drastically inhibited intrinsic electron transfer from CuA to heme a. This intrinsic rate is biphasic; the proportion that is slow is pH dependent, as is the relative intensity of the two EPR signal components. At pH 8, the heme a EPR signal at 2.72 is most intense, and the electron transfer rate (CuA to heme a) is 10-130 s(-1), compared to wild-type at 90,000 s(-1). At pH 5.5, the signal at 2.78 is intensified, and a biphasic rate is observed, 50% fast (approximately wild type) and 50% slow (90 s(-1)). The data support the prediction that the hydrogen-bonding partner of the histidine ligand of heme a is one determinant of the EPR spectral difference between bovine and bacterial CcO. We further demonstrate that the heme a redox potential can be dramatically altered by a nearby carboxyl, whose protonation leads to a proton-coupled electron transfer process.     

Lactic acid production from hemicellulosic hydrolyzate by cells of <Emphasis Type="Italic">Lactobacillus bifermentans</Emphasis> immobilized in Ca-alginate using response surface methodology

Consumption of hexoses/pentoses and production of lactic acid by Lactobacillus bifermentans were investigated in optimized culture medium and hemicellulosic hydrolyzates. The hydrolyzate used had the following composition (expressed in gL⁻¹): xylose 50 ± 5 gL⁻¹; glucose 18 ± 3 gL⁻¹; arabinose 29 ± 5 gL⁻¹. The immobilization experiments were conducted with microbial cells entrapped in calcium alginate beads. The results indicate that maximum concentrations of lactic acid were produced after 54 h of fermentation. All glucose and arabinose in wheat bran hydrolyzate were consumed during fermentation. Only xylose was not completely consumed. The substrate consumption rate was 3.2 gh⁻¹, 1.9 gh⁻¹, 1.6 gh⁻¹ respectively for glucose, arabinose, and xylose. The optimized culture condition gave a lactic acid concentration and metabolic yield of 62.77 gL⁻¹ and 0.83 gg⁻¹. These parameters improved to 41.3 gL⁻¹ and 0.47 gg⁻¹ respectively, when cell free was used.     

Enumeration of micronucleated CD71-positive human reticulocytes with a single-laser flow cytometer

The extreme rarity of micronucleated reticulocytes (RETs) in the peripheral blood of non-splenectomized humans has precluded facile enumeration of these cells, as well as evaluation of this endpoint as an index of cytogenetic damage. In this report, we describe a high-throughput, single-laser flow cytometric system for scoring the incidence of micronuclei (MN) in newly formed human RETs. The procedure is based on an immunochemical reagent that differentially labels the most immature fraction of RETs from mature erythrocytes based on the expression level of the transferrin receptor (also known as CD71). The resolution of four erythrocyte populations (young RETs and mature erythrocytes, with and without MN) was achieved for human blood cells treated with phycoerythrin-conjugated anti-CD71, RNase, and either SYTOX Green or SYBR Green I nucleic acid dyes. Anti-glycophorin A labeling of erythroid cells (CyChrome conjugate) was also incorporated into the staining procedure to ensure that debris or other potential artifacts did not adversely impact the analyses. Instrument calibration procedures utilizing malaria-infected rodent erythrocytes were also developed, and are described. Using this analytical system, blood samples from 10 healthy non-splenectomized human volunteers were analyzed for micronucleus frequencies with a single-laser flow cytometer. Average micronucleus frequencies in the mature and most immature fraction of RETs were 0.016 and 0.19%, respectively. Blood samples from three healthy splenectomized volunteers were also evaluated. As expected, these samples exhibited higher micronucleus frequencies in the mature subset of erythrocytes (range 0.03-0.18%). The resulting data suggest that MN can be quantified in human erythrocyte populations with a single-laser flow cytometer, and that the frequency of MN cells in the youngest reticulocyte population approaches values expected in the absence of splenic selection against MN-erythrocytes. This high throughput system is potentially important for evaluating the value of the micronucleated reticulocyte endpoint as an index of chromosome breakage and/or chromosome segregational abnormalities in human populations.     

A new Saccharomyces cerevisiae strain with a mutant Smt3-deconjugating Ulp1 protein is affected in DNA replication and requires Srs2 and homologous recombination for its viability

The Saccharomyces cerevisiae Srs2 protein is involved in DNA repair and recombination. In order to gain better insight into the roles of Srs2, we performed a screen to identify mutations that are synthetically lethal with an srs2 deletion. One of them is a mutated allele of the ULP1 gene that encodes a protease specifically cleaving Smt3-protein conjugates. This allele, ulp1-I615N, is responsible for an accumulation of Smt3-conjugated proteins. The mutant is unable to grow at 37 degrees C. At permissive temperatures, it still shows severe growth defects together with a strong hyperrecombination phenotype and is impaired in meiosis. Genetic interactions between ulp1 and mutations that affect different repair pathways indicated that the RAD51-dependent homologous recombination mechanism, but not excision resynthesis, translesion synthesis, or nonhomologous end-joining processes, is required for the viability of the mutant. Thus, both Srs2, believed to negatively control homologous recombination, and the process of recombination per se are essential for the viability of the ulp1 mutant. Upon replication, mutant cells accumulate single-stranded DNA interruptions. These structures are believed to generate different recombination intermediates. Some of them are fixed by recombination, and others require Srs2 to be reversed and fixed by an alternate pathway.