Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry

The septins are a conserved family of GTP-binding proteins that, in the baker''s yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. Surprisingly, we detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in alpha-factor arrested that do not display an assembled septin structure. Furthermore, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified.Complementary methods like SPLIFF and immunoprecipitation allowed us to more exactly define the spatial and temporal characteristics of selected hits of the AP-MS screen.     

Specificity of Collybistin-Phosphoinositide Interactions: IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS*

The regulatory protein collybistin (CB) recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2_SH3+) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally “opens” CB2_SH3+ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering.     

The role of phospholipase A activity in rat liver microsomal lipid peroxidation

The involvement of phospholipase(s) A in lipid peroxidation of rat liver microsomes was investigated by: (a) determining the effects of phospholipase A inhibitors (p-bromophenylacyl bromide, chlorpromazine, mepacrine) on the accumulation of thiobarbituric acid reactivity or on levels of oxidized phospholipids in response to selected oxidative stimuli and (b) measurement of phospholipase A activities in response to these agents. Lipid peroxidation in response to various peroxidation systems was inhibited completely by exposure of microsomes to p-bromophenylacyl bromide (250 microM). The effectiveness of p-bromophenylacyl bromide was dependent on the presence of glutathione (200 microM) in preincubation mixtures. Chlorpromazine (100 microM) and mepacrine (100 microM) also effectively inhibited peroxidation, and their potency was independent of glutathione. The accumulation of oxidized phospholipids in response to the potent peroxidation stimulus alloxan/ferrous ion was similarly inhibited by p-bromophenylacyl bromide, although the level of oxidized phospholipid in response to the initiator ADP/ferrous ion was not affected. Microsomal phospholipase A1 activity, assessed using a liposomal substrate, was substantially enhanced by promoters of lipid peroxidation. Phospholipase A2 activity was not detected using a liposomal substrate but was evident using radiolabeled microsomes as endogenous substrate and was enhanced by oxidative stimuli. We conclude that phospholipase A activity may play an integral role in the microsomal lipid peroxidation mechanism. Based on this study, we hypothesize a role for phospholipases in facilitating propagation reactions.     

Effects of calcium channel blockers and DCMU on motility and the photophobic response of Gyrodinium dorsum

The effects of the calcium channel blockers, verapamil, diltiazem and lanthanum ions and the Ca²⁺ dependency on motility as well as the photophobic response (stop-response) of Gyrodinium dorsum were studied. At Ca²⁺ concentrations below 10^-3 M, motility was inhibited. La³⁺ inhibits the stop-response, in contrast to verapamil and diltiazem. The only calcium channel blocker that increased the amount of non-motile cells was verapamil. The results indicate that motility are Ca²⁺ dependent and that the stop-responses of G. dorsum could be affected by extracellular Ca²⁺. Effects of the photosythesis inhibitor (DCMU) on the stop-response was also determined. With background light of different wavelength (614, 658 and 686 nm) the stop-response increased. DCMU inhibited this effect of background light. Negative results with the monoclonal antibody Pea-25 directed to phytochrome and the results with DCMU, indicate that the stop-response of G. dorsum is coupled to photosynthesis rather than to a phytochrome-like pigment. Oxygen evolution, but not cell movement, was completely inhibited by 10^-6 M DCMU.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-methylurea - DILT diltiazem - DMSO dimethylsulfoxide - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - VER verapamil     

Increased therapeutic efficacy of zidovudine in combination with vitamin E

Antiviral activity and bone marrow toxicity of 3'-azido-3'deoxythymidine (Zidovudine; AZT) was evaluated in the presence of alpha-D-tocopherol acid succinate (ATS) in the MT4 cell line and in murine hematopoietic progenitor cells, respectively. At varying concentrations (.016 to .125 microM) of AZT, addition of ATS (5 to 15 micrograms/ml) showed a dose-dependent increase in anti-HIV activity. The ED90 of AZT in this test system was 0.37 microM, whereas in the presence of ATS (15 micrograms/ml) it was 0.06 microM, thus producing an approximately 6-fold increase in anti-HIV activity. In contrast, in murine bone marrow cells, ATS (4 micrograms/ml) showed significant protection (p less than 0.05) against AZT-induced toxicity as measured by CFU-E and CFU-GM assays. The IC50 values in the presence and absence of ATS for CFU-E were 3.7 and 1.5 microM, whereas for CFU-GM were 6.0 and 2.7 microM, respectively. Overall, these data suggest that AZT in combination with ATS has greater therapeutic efficacy against HIV-1.     

Characteristic chromosome abnormalities,including rearrangements of 6p,del(7q), +12, and t(12;14), in 44 uterine leiomyomas

Summary The cytogenetic analysis of 224 leiomyomas from 138 patients is presented. An insufficient number of mitoses was found in 35 tumors, normal karyotypes in 145, and clonal chromosome aberrations were detected in 44. The three previously identified cytogenetic subgroups were all represented in this series: del(7) (q21.2q31.2) was found in 11, trisomy 12 in five, and t(12;14)(q14-15;q23-24) in one leiomyoma. Rearrangements of 6p, including deletions, inversions, and various translocations, were found in eight tumors, thus delineating a new cytogenetic subgroup of uterine leiomyoma. The remaining 21 karyotypically abnormal tumors had nonrecurrent changes. One leiomyoma had two cytogenetically unrelated clones characterized by del(7)(q21.2 q31.2) and +12. Karyotypic changes in two separate leiomyomas from the same uterus were identified in five patients; in three of them, different anomalies were found in the two tumors, whereas cytogenetically identical aberrations – del(7q) and dic(21;22) – were detected in two macroscopically discrete tumors. These findings suggest that whereas some multiple leiomyomas originate independently, others may be derived from the same neoplastic clone.     

Images of purple membrane at 2.8 A resolution obtained by cryo-electron microscopy

Improvements in technique have produced electron micrographs of purple membrane that provide, after computer analysis, reproducibly measurable diffraction peaks extending to 2.8 A (1 A = 0.1 nm). The improvements include better specimen preparation, a more stable cryo-electron microscope with better alignment and the addition of an image-processing step, which gives weights to local areas of the image according to the local strength of the periodic component of the image. These improvements have enabled the calculation of a directly phased projection map at 2.8 A resolution.     

Cholinesterase Activities in Uterus of Normal and Fenchlorphos Treated Blue Foxes (Alopex Lagopus) during Various Reproductive States

The uterine acetylcholinesterase and total cholinesterase (acetylcholinesterase plus butyrylcholinesterase) activities in normal and fenchlorphos treated blue fox vixens were determined during various reproductive states. AChE and Total-ChE of non-medicated vixens in oestrus were about one half of those in anoestrus. In pregnant uteri (luteal phase) the activities were 25 % and 30% compared to anoestrus. In vixens given 100 mg/kg fenchlorphos for 3 weeks during anoestrus, the remaining activity of AChE in uterus were in average 37%. Pregnant and non-pregnant vixens in the luteal phase medicated prior to mating and during time of implantation, displayed AChE activities which were only moderabely reduced (remaining activities 83% and 72% compared to medicated animals in anoestrus: remaining activity 37%). Plasma ChE-activity increased during pregnancy in the controls while enzyme activity was strongly reduced in animals given 100 mg/kg fenchlorphos daily through the whole pregnancy. It was concluded that the previous reported embryotoxic effect of fenchlorphos in the blue fox did not seem to be directed towards the moderate inhibition of the uterine cholinesterases.