Metabolic Stability of Hippocampal Slice Preparations During Prolonged Incubation

Abstract: Hippocampal slices were prepared under three conditions: (1) in medium containing glucose and oxygen at 4°C; (2) as in (1), but at 37°C; (3) in medium devoid of glucose and oxygen at 37°C. The rates of recovery to roughly steady-state levels and through 8 h of incubation were monitored for energy metabolite levels and related parameters. In vitro stable values are compared with in situ hippocampal levels. Regardless of the conditions under which slices were prepared, metabolite levels required up to 3 h to stabilize, and these levels were maintained or improved through 8 h of incubation. Further, the maximal concentrations of metabolites were independent of the conditions of slice preparation. Total adenylates and total creatine levels reached 55% of those in vivo. Lactate decreased from the decapitation-induced high levels, but stabilized at concentrations about twice those in rapidly frozen brain. Cyclic AMP and cyclic GMP exhibited peak levels at 30 min of incubation, and cyclic GMP remained elevated for 3 h. Although all three methods of slice preparation resulted in similar metabolite profiles on incubation, the initial decreases in high energy phosphates were delayed by chilling. Most striking, the slices prepared in the absence of glucose and oxygen exhibited much smaller orthodromic evoked potentials in the dentate gyrus. The presence of glucose and oxygen during preparation of the slices appears to be critical to the electrophysiological response of the tissue.     

cDNA cloning of translationally controlled tumor protein/histamine releasing factor (TCTP/HRF) from the intertidal harpacticoid copepod Tigriopus japonicus.

We synthesized a cDNA library from the intertidal copepod Tigriopus japonicus, converted it to phagemids and sequenced expressed sequence tags (ESTs). Of these, Tigriopus translationally controlled tumor protein/histamine releasing factor (TCTP/HRF) was further characterized. The Tigriopus TCTP/HRF gene encoded 172 amino acid residues and showed high similarity to Drosophila but moderate similarity to other annelids (e.g. Brugia, Wuchereria and C. elegans). The Tigriopus TCTP/HRF gene appeared in the same clade as the annelids. Here, we describe the analysis of the Tigriopus TCTP/HRF gene.     

The conserved ubiquitin-like protein Hub1 plays a critical role in splicing in human cells

Different from canonical ubiquitin-like proteins, Hub1 does not form covalent conjugates with substrates but binds proteins noncovalently. In Socchoromyces cerevisioe, Hub1 associates with spUceosomes and mediates alternative splicing of SRCI, without affecting pre-mRNA splicing generaity. Human Hub1 is highty similar to its yeast homotog, but its cellular function remains largely unexplored. Here, we show that human Hub1 binds to the spliceosomal protein Snu66 as in yeast; however, unlike its 5. cerevisioe homolos, human Hub1 is essential for viability. Prolonged in vivo depletion of human Hub1 leads to various cellular defects, including splicing speckle abnormalities, partial nuclear retention of mRNAs, mitotic catastrophe, and consequently cell death by apoptosis. Early consequences of Hub1 depletion are severe splicing defects, however, only for specific splice sites leading to exon skipping and intron retention. Thus, the ubiquitin-iike protein Hub1 is not a canonlcal spliceosomal factor needed generally for splicing, but rather a modulator of spliceosome performance and facilitator of alternative splicing.     

Specific Interaction of the Unmodified Bacteriocin Lactococcin 972 with the Cell Wall Precursor Lipid II

Lactococcin 972 (Lcn972) is a nonlantibiotic bacteriocin that inhibits septum biosynthesis in Lactococcus lactis rather than forming pores in the cytoplasmic membrane. In this study, a deeper analysis of the molecular basis of the mode of action of Lcn972 was performed. Of several lipid cell wall precursors, only lipid II antagonized Lcn972 inhibitory activity in vivo. Likewise, Lcn972 only coprecipitated with lipid II micelles. This bacteriocin inhibited the in vitro polymerization of lipid II by the recombinant S. aureus PBP2 and the addition to lipid II of the first glycine catalyzed by FemX. These experiments demonstrate that Lcn972 specifically interacts with lipid II, the substrate of both enzymes. In the presence of Lcn972, nisin pore formation was partially hindered in whole cells. However, binding of Lcn972 to lipid II could not compete with nisin in lipid II-doped 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes, possibly indicating a distinct binding site. The existence of a putative cotarget for Lcn972 activity is discussed in the context of its narrow inhibitory spectrum and the localized action at the division septum. To our knowledge, this is the first unmodified bacteriocin that binds to the cell wall precursor lipid II.