Characterization of novel SF3b and 17S U2 snRNP proteins,including a human Prp5p homologue and an SF3b DEAD-box protein

Mass spectrometry was used to identify novel proteins associated with the human 17S U2 snRNP and one of its stable subunits, SF3b. Several additional proteins were identified, demonstrating that 17S U2 snRNPs are significantly more complex than previously thought. Two of the newly identified proteins, namely the DEAD-box proteins SF3b125 and hPrp5 (a homologue of Saccharomyces cerevisiae Prp5p) were characterized further. Immunodepletion experiments with HeLa nuclear extract indicated that hPrp5p plays an important role in pre-mRNA splicing, acting during or prior to prespliceosome assembly. The SF3b-associated protein SF3b125 dissociates at the time of 17S U2 formation, raising the interesting possibility that it might facilitate the assembly of the 17S U2 snRNP. Finally, immunofluorescence/FISH studies revealed a differential subnuclear distribution of U2 snRNA, hPrp5p and SF3b125, which were enriched in Cajal bodies, versus SF3b155 and SF3a120, which were not; a model for 17S U2 snRNP assembly based on these findings is presented. Taken together, these studies provide new insight into the composition of the 17S U2 snRNP and the potential function of several of its proteins.     

Assessment of stallion spermatozoa viability by flow cytometry and light microscope analysis

Viability of spermatozoa can be assessed by numerous methods, but many are slow and poorly repeatable, and subjectively assess only 100 to 200 spermatozoa per ejaculate. We collected two ejaculates from each of 4 stallions, and extended them to 50x10(6) sperm/mL in a nonfat dried milk solids glucose extender (EZ Mixin). Half the ejaculate was freeze-killed by immersing in liquid nitrogen for 10 min. Aliquots using appropriate volumes of live and freeze-killed spermatozoa provided the following ratios of live:dead spermatozoa: 100:0, 75:25, 50:50, 25:75, 0:100. We determined the viability of each aliquot by 1) motility; 2) eosin-nigrosin staining; and 3) dual fluorescent staining. For the latter, aliquots incubated with SYBR-14 and propidium iodide had live and dead spermatozoa quantitated by fluorescent microscope (2 x 100 sperm/sample) and flow cytometry (10,000 sperm/sample). We found a linear relationship between the ratio of live:dead spermatozoa and the percentage of spermatozoa counted as live (P<0.0001). For fresh spermatozoa, correlation coefficients of the known live:dead ratio were high for all methods (eosin-nigrosin, r>0.75; fluorescent microscope, r>0.76; flow cytometry, r>0.75; motility, r>0.76). To determine viability of cryopreserved equine spermatozoa, we froze 17 fresh ejaculates from 6 stallions in a glycine extender. Each sample was thawed, extended 1:1 with EZ Mixin and evaluated as above. Cryopreserved spermatozoa assessed by flow cytometry tended to be less well correlated (r<0.68) with the other methods, and estimates were significantly higher with eosin-nigrosin staining (P<0.001). This study shows that different methods may equally estimate viability of fresh equine spermatozoa. However, evaluation by flow cytometry appears to be less precise with cryopreserved spermatozoa.     

TPX2, A novel xenopus MAP involved in spindle pole organization

TPX2, the targeting protein for Xenopus kinesin-like protein 2 (Xklp2), was identified as a microtubule-associated protein that mediates the binding of the COOH-terminal domain of Xklp2 to microtubules (Wittmann, T., H. Boleti, C. Antony, E. Karsenti, and I. Vernos. 1998. J. Cell Biol. 143:673-685). Here, we report the cloning and functional characterization of Xenopus TPX2. TPX2 is a novel, basic 82.4-kD protein that is phosphorylated during mitosis in a microtubule-dependent way. TPX2 is nuclear during interphase and becomes localized to spindle poles in mitosis. Spindle pole localization of TPX2 requires the activity of the dynein-dynactin complex. In late anaphase TPX2 becomes relocalized from the spindle poles to the midbody. TPX2 is highly homologous to a human protein of unknown function and thus defines a new family of vertebrate spindle pole components. We investigated the function of TPX2 using spindle assembly in Xenopus egg extracts. Immunodepletion of TPX2 from mitotic egg extracts resulted in bipolar structures with disintegrating poles and a decreased microtubule density. Addition of an excess of TPX2 to spindle assembly reactions gave rise to monopolar structures with abnormally enlarged poles. We conclude that, in addition to its function in targeting Xklp2 to microtubule minus ends during mitosis, TPX2 also participates in the organization of spindle poles.     

Identification of amino acid residues of GABA(A) receptor subunits contributing to the formation and affinity of the tert-butylbicyclophosphorothionate binding site

A chimeric GABA(A) receptor subunit was constructed that contained the beta3 sequence from the N-terminus to the first two amino acids of the second transmembrane (TM2) domain. The remaining part of this chimera had the sequence of the alpha1 subunit. On co-expression with alpha1 subunits, this chimera was able to form heterooligomeric channels that were open in the absence of GABA. Picrotoxin and tert-butylbicyclophosphorothionate (TBPS) were able to block these channels with low potency. These channels exhibited high-affinity [3H]muscimol but no high-affinity [35S]TBPS binding sites. Introduction of V251, A252, and L253 of the beta3 subunit into the chimera resulted in the formation of closed channels that could be opened by GABA. The introduction of A252 and L253 of the beta3 subunit into this chimera was sufficient to reconstitute the specific high-affinity [35S]TBPS binding site in receptors composed of the chimera and alpha1 subunits. Replacement of other amino acids of the TM2 region of the chimera with corresponding amino acids of the beta3 subunit modulated the affinity of this [35S]TBPS binding site. Results obtained provide important information on the structure-function relationship of GABA(A) receptors.     

The sec14 homology module of neurofibromin binds cellular glycerophospholipids: mass spectrometry and structure of a lipid complex

Neurofibromin is the protein product of the tumor suppressor gene NF1, alterations of which are responsible for the pathogenesis of the common disorder Neurofibromatosis type I (NF1). The only well-characterized function of neurofibromin is its RasGAP activity, contained in the central GAP related domain (GRD). By solving the crystal structure of a 31 kDa fragment at the C-terminal end of the GRD we have recently identified a novel bipartite lipid-binding module composed of a Sec14 homologous and a previously undetected pleckstrin homology (PH)-like domain. Using lipid exchange assays along with mass spectrometry we show here that the Sec14-like portion binds to 1-(3-sn-phosphatidyl)-sn-glycerol (PtdGro), (3-sn-phosphatidyl)-ethanolamine (PtdEtn) and -choline (PtdCho) and to a minor extent to (3-sn-phosphatidyl)-l-serine (PtdSer) and 1-(3-sn-phosphatidyl)-d-myo-inositol (PtdIns). Phosphorylated PtdIns (PtdInsPs) are not detected as binders in the mass spectrometry assay, but their soluble inositol-phosphate headgroups and related compounds can inhibit the lipid exchange reaction. We also present here the crystal structure of this module with the Sec14 portion bound to a cellular glycerophospholipid ligand. Our structure has model character for the substrate-bound form of yeast Sec14p, of which only detergent bound structures are available so far. To assess potential regulation of the lipid exchange reaction in detail, we present a novel strategy using nanospray mass spectrometry. Ion intensities of initial phospholipids and exchanged deuterated analogues bound by the protein module allow the quantitative analysis of differences in the exchange activity under various conditions.     

Preparation of tetrahydroimidazo[2,1-a]isoquinolines and their use as inhibitors of gastric acid secretion

A series of novel tetrahydroimidazo[2,1-a]isoquinolines was prepared based on a hetero Diels–Alder reaction between an enamine and 1,2,4-triazine as key step. A structure–activity relationship was established focussing on the influence of the substitution pattern in position 3 and 6 of the heterocycle on antisecretory activity, lipophilicity, and pK_a value. Potent inhibitors of the gastric acid pump were identified.