Aggregation-dependent turnover of flagellar adhesion molecules in chlamydomonas gametes

Previous studies on flagellar adhesion in chlamydomonas (Snell, W. and S. Roseman. 1979. J. Biol. Chem. 254:10820-10829.) have shown that as gametes adhere to flagella isolated from gametes of the opposite mating type, the adhsiveness of the added flagella but not of the gametes is lost. The studies reported here show that the addition of protein synthesis inhibitors (cycloheximide [CH] or anisomycin) to the medium of such cell- flagella mixtures causes the cells to lose their adhesiveness. This loss, however, occurs only after the cells have interacted with 4-8 flagella/cell and does not occur if the cells are kept in CH (7 h) without aggregating. The availability of an impotent (imp) mating type plus (MT(+)) mutant (provided by U.W. Goodenough), which adheres but is unable to undergo the fusion that normally follows adhesion, made it possible to determine whether a similar loss of adhesiveness occurs in mixtures of matting type minus (mt(-)) and imp mt(+) gametes. In the absence of inhibitor, mt(-) and imp mt(+) gametes adhered to each other (without fusing) for several hours; however, in the presence of CH or anisomycin, the gametes began to de-adhere 35 min after mixing, and, by 90 min, 100 percent of the cells were single again. This effect was reversible, and the rapid turnover of cells were single again. This effect was reversible, and the rapid turnover of molecules involved in adhesion occurred only during adhesion inasmuch as gametes pretreated for 4 h with CH were able to aggregate in CH for the same length of time as nonpretreated cells aggregated in CH. By the addition of CH at various times after the mt(-) and imp mt(+) gametes were mixed, measurements were made of the “pool size” of the molecules involved in adhesion. The pool reached a minimum after 25 min of aggregation, rapidly increased for the next 25 min, and then leveled off at the premixing level. These results suggest that flagellar adhesion in chlamydomonas causes modification of surface molecules (receptors, ligands), which brings about their inactivation and stimulates their replacement.     

Angelman syndrome and severe infections in a patient with de novo 15q11.2–q13.1 deletion and maternally inherited 2q21.3 microdeletion

Angelman syndrome is a neurodevelopmental disorder characterized by mental retardation, severe speech disorder, facial dysmorphism, secondary microcephaly, ataxia, seizures, and abnormal behaviors such as easily provoked laughter. It is most frequently caused by a de novo maternal deletion of chromosome 15q11–q13 (about 70–90%), but can also be caused by paternal uniparental disomy of chromosome 15q11–q13 (3–7%), an imprinting defect (2–4%) or in mutations in the ubiquitin protein ligase E3A gene UBE3A mostly leading to frame shift mutation. In addition, for patients with overlapping clinical features (Angelman-like syndrome), mutations in methyl-CpG binding protein 2 gene MECP2 and cyclin-dependent kinase-like 5 gene CDKL5 as well as a microdeletion of 2q23.1 including the methyl-CpG binding domain protein 5 gene MBD5 have been described. Here, we describe a patient who carries a de novo 5 Mb-deletion of chromosome 15q11.2–q13.1 known to be associated with Angelman syndrome and a further, maternally inherited deletion 2q21.3 (~ 364 kb) of unknown significance. In addition to classic features of Angelman syndrome, she presented with severe infections in the first year of life, a symptom that has not been described in patients with Angelman syndrome. The 15q11.2–q13.1 deletion contains genes critical for Prader–Willi syndrome, the Angelman syndrome causing genes UBE3A and ATP10A/C, and several non-imprinted genes: GABRB3 and GABRA5 (both encoding subunits of GABA A receptor), GOLGA6L2, HERC2 and OCA2 (associated with oculocutaneous albinism II). The deletion 2q21.3 includes exons of the genes RAB3GAP1 (associated with Warburg Micro syndrome) and ZRANB3 (not disease-associated). Despite the normal phenotype of the mother, the relevance of the 2q21.3 microdeletion for the phenotype of the patient cannot be excluded, and further case reports will need to address this point.     

Descriptions of new taxa of the Clausiliidae from Turkey (Mollusca: Stylommatophora)

Summary

Several new taxa of the Clausiliidae were described From Turkey: Dobatia multidentifera n. sp., Albinaria monocristata n. sp., Albinaria basalifera n. sp., Roseniella (Chavchetia) n. subgen., Roseniella (Chavchetia) rufina n. sp., Armenica euprepes truncata n. ssp., Armenica multispirata n. sp., Armenica bicarinata grisea n. ssp., Kazancia n. gen., and Kazancia monticola n. sp. Investigations were made of the genital morphology of some species, and the arrangement of the taxa in Nordsieck’s classification (H, NORDSIECK 1979) is discussed.     

Consequences of long-term selenium-deficient diet on the prostacyclin and thromboxane release from rat aorta

It is known that peroxides, which are increased during Se deficiency because of reduced glutathione peroxidase (GSH-Px) activity, can influence the prostacyclin I₂/thromboxane A₂ (PGI₂/TXA₂) ratio. In this study we analyzed the PGI₂ and TXA₂ formation of aortas of long-term Se-deficient rats. Despite low GSH-Px activity in the Se-deficient group, the basal PGI₂ and TXA₂ formation was not different versus control animals (PGI₂: 2295 ± 1134 pg/mg vs 2940 ± 1134 pg/mg; TXA₂: 3.83 ± 1.06 pg/mg vs 5.67 ± 2.99 pg/mg). However, we checked the capacity of the aortas of Se-deficient rats to compensate for a suddenly increased peroxide concentration. After peroxide stimulation, the PGI₂ release was significantly lower in the Se-deficient group compared to the control group (PGI₂: 3507 ± 1829 pg/mg vs 7986 ± 2636 pg/mg). Again, the TXA₂ release did not show any differences. The release ratio of PGI₂/TXA₂ decreased under peroxide stress in Se-deficient animals. Although long-term Se deficiency showed a relatively well-balanced metabolism under resting conditions, sudden stress, accompanied by an excessive radical production, cannot be compensated.     

Mass spectrometric analysis of GAP-43/neuromodulin reveals the presence of a variety of fatty acylated species

GAP-43 (neuromodulin) is a protein kinase C substrate that is abundant in developing and regenerating neurons. Thioester-linked palmitoylation at two cysteines near the GAP-43 N terminus has been implicated in directing membrane binding. Here, we use mass spectrometry to examine the stoichiometry of palmitoylation and the molecular identity of the fatty acid(s) attached to GAP-43 in vivo. GAP-43 expressed in either PC12 or COS-1 cells was acetylated at the N-terminal methionine. Approximately 35% of the N-terminal GAP-43 peptides were also modified by palmitate and/or stearate on Cys residues. Interestingly, a variety of acylated species was detected, in which one of the Cys residues was acylated by either palmitate or stearate, or both Cys residues were acylated by palmitates or stearates or a combination of palmitate and stearate. Depalmitoylation of membrane-bound GAP-43 did not release the protein from the membrane, implying that additional forces function to maintain membrane binding. Indeed, mutation of four basic residues within the N-terminal domain of GAP-43 dramatically reduced membrane localization of GAP-43 without affecting palmitoylation. These data reveal the heterogeneous nature of S-acylation in vivo and illustrate the power of mass spectrometry for identification of key regulatory protein modifications.     

Invertase in Immobilized Cells of Eschscholtzia Californica

Cell suspension culture of Eschscholtzia californica Cham. were permeabilized by Tween 20 or 80, and immobilized by glutaraldehyde. The highest invertase activity was at pH 4.5 and temperature 50 °C. The hydrolysis of the substrate was linear for 5 h reaching 60 % conversion. The cells had high invertase activity and a good stability, and in long-term storage they showed convenient physico-mechanical properties.     

Transcriptional regulation of cytosol and membrane alanyl-aminopeptidase in human T cell subsets

Aminopeptidase inhibitors strongly affect the proliferation and function of immune cells in man and animals and are promising agents for the pharmacological treatment of inflammatory or autoimmune diseases. Membrane alanyl-aminopeptidase (mAAP) has been considered as the major target of these anti-inflammatory aminopeptidase inhibitors. Recent evidence also points to a role of the cytosol alanyl-aminopeptidase (cAAP) in the immune response. In this study we used quantitative RT-PCR to determine the mRNA expression of both cAAP and mAAP in resting and activated peripheral T cells and also in CD4+, CD8+, Th1, Th2 and Treg (CD4+ CD25+) subpopulations. Both mAAP and cAAP mRNAs were expressed in all cell types investigated, and in response to activation their expression appeared to be upregulated in CD8+ cells, but downregulated in Treg cells. In CD4+ cells, mAAP and cAAP mRNAs were affected in opposite ways in response to activation. The cAAP-specific inhibitor, PAQ-22, did not affect either cAAP or mAAP expression in activated CD4+ or CD8+ cells, whereas in activated Treg cells it markedly upregulated the mRNA levels of both aminopeptidases. The non-discriminatory inhibitor, phebestin, significantly increased the amount of mAAP and cAAP mRNA in CD4+ and that of cAAP in Treg cells.     

Effect of paracetamol (acetaminophen) and ibuprofen on body temperature in acute ischemic stroke PISA, a phase II double-blind, randomized, placebo-controlled trial [ISRCTN98608690]

Background

Body temperature is a strong predictor of outcome in acute stroke. In a previous randomized trial we observed that treatment with high-dose acetaminophen (paracetamol) led to a reduction of body temperature in patients with acute ischemic stroke, even when they had no fever. The purpose of the present trial was to study whether this effect of acetaminophen could be reproduced, and whether ibuprofen would have a similar, or even stronger effect.

Methods

Seventy-five patients with acute ischemic stroke confined to the anterior circulation were randomized to treatment with either 1000 mg acetaminophen, 400 mg ibuprofen, or placebo, given 6 times daily during 5 days. Treatment was started within 24 hours from the onset of symptoms. Body temperatures were measured at 2-hour intervals during the first 24 hours, and at 6-hour intervals thereafter.

Results

No difference in body temperature at 24 hours was observed between the three treatment groups. However, treatment with high-dose acetaminophen resulted in a 0.3°C larger reduction in body temperature from baseline than placebo treatment (95% CI: 0.0 to 0.6 °C). Acetaminophen had no significant effect on body temperature during the subsequent four days compared to placebo, and ibuprofen had no statistically significant effect on body temperature during the entire study period.

Conclusions

Treatment with a daily dose of 6000 mg acetaminophen results in a small, but potentially worthwhile decrease in body temperature after acute ischemic stroke, even in normothermic and subfebrile patients. Further large randomized clinical trials are needed to study whether early reduction of body temperature leads to improved outcome.     

Bioadhesion of supramolecular structures at supported planar bilayers as studied by the quartz crystal microbalance

A quartz crystal microbalance (QCM) was used to study the adhesion behavior of supramolecular aggregates at supported planar bilayers (SPBs). The QCM technique is a suitable method to detect the adsorption of biomolecules at the quartz surface owing to its sensitivity for changes in mass and viscoelastic properties. To simulate biomembranes, the quartz plates were coated with highly ordered lipid films. Therefore, a combination of self-assembled monolayers and Langmuir-Blodgett films was used. Firstly, the adsorption of liposomes coupled with the lectin concanavalin A was investigated at glycolipid-containing model membranes. Using different carbohydrates, it was possible to determine specific and nonspecific parts of the interactions. The adhesion occurred owing to specific lectin-carbohydrate interactions (about 20%) and to nonspecific interactions (about 80%). The composition of the liposomes was changed to simulate the structure of a native biomembrane consisting of the glycocalix, the lipid-protein bilayer, and the cytoskeleton. An artificial glycocalix was created by incorporating poly(ethylene glycol) into the liposomes. Liposomes which were intravesicular polymerized with polyacrylamide or polyacrylcholate simulated the cytoskeleton. It was determined that the modified liposomes had significant lower interactions with SPBs. The adsorption was reduced by approximately 80% compared to unmodified liposomes. Secondly, a model was developed for the detection of interactions between simple or mixed bile salt micelles and model membranes. It was found that simple bile salts did not adsorb at model membranes. Binary systems consisting of bile salt and phospholipid induced only small interactions. On the other hand, ternary systems consisting of bile salt, phospholipid, and fatty acid showed strong interactions. A dependence on the chain length of the fatty acid was observed. Thirdly, the interaction between ganglioside-containing model membranes and cholera toxin (beta-subunit) was investigated. Different ganglioside fractions showed varying adsorption in the following sequence: GM1 > GD1a > GD1b > GT1b.