Internalization, recycling, and redistribution of vasopressin receptors in rat hepatocytes

Three hours after isolation, cultured hepatocytes have approximately 150,000 surface vasopressin receptors/cell, and these exhibit a Kd for 125I-vasopressin of 6 nM based on calculation of Koff/Kon, or a Kd of 9.5 nM based on Scatchard plot analysis. After the binding of 125I-vasopressin to its receptor on the hepatocyte surface, this complex is internalized with a t1/2 of 3-6 min. Following this internalization, the number of vasopressin receptors on the cell surface is restored both in vitro and in the isolated perfused liver with a t1/2 of 8-10 min. This restoration is blocked in vitro by incubation of the hepatocytes at 18 degrees C, but not by cycloheximide, suggesting that internalized vasopressin receptors recycle back to the cell surface. Prolonged incubation of hepatocytes with vasopressin results in the loss of greater than 75% of the vasopressin surface binding at concentrations of vasopressin approximately equivalent to its Kd. The binding of vasopressin to cultured hepatocytes 3-5 h after isolation resembles binding to the isolated perfused whole liver with respect to receptor dynamics. During culture for 48 h, however, we observe a progressive loss of hepatocyte surface vasopressin receptors. Concomitant with this reduction in surface receptors with time in culture, there appears to be a marked elevation in intracellular receptors.     

Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18‐1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho‐proteomics abolished the stimulatory effect of Munc18‐1 on SNARE complex formation (“SNARE‐templating”) and membrane fusion in vitro. Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18‐1‐null neurons expressing Munc18‐1_Y473D. Synaptic transmission was temporarily restored by high‐frequency stimulation, as well as by a Munc18‐1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non‐phosphorylatable Munc18‐1 supported normal synaptic transmission. We propose that SFK‐dependent Munc18‐1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post‐docking SNARE‐templating role of Munc18‐1, resulting in a largely abolished pool of releasable synaptic vesicles.     

铁皮石斛的离体开花

铁皮石斛(Dendrobium candidum),为一种野生兰科植物,在栽培条件下,从种子萌发到开花通常需要3～4a.研究了多种植物激素和多胺对该种石斛组织培养中花芽形成的影响,结果表明在培养基中加入合适浓度的亚精胺(spermidine)或BA(6-苄基腺嘌呤),或同时加入NAA(萘乙酸)和BA均可诱导原球茎或由之形成的无根小苗在3～6个月开花,频率在31.6%～45.8%.当将原球茎在加有ABA(脱落酸)的培养基上预培养后再移到加有BA的培养基上,花芽形成的频率可提高到平均达82.8%(个别实验中可达100%),这种诱导提早开花的现象也与实验材料的发育阶段(原球茎、无根小苗、已生根的小苗)有关,通常发生在根的形成受到完全或部分抑制的情况中.     

ConA激活小鼠胸腺T淋巴细胞增殖过程中c-myc与核骨架蛋白的结合

采用DNA-蛋白质体外吸附的方法研究伴刀豆球蛋白激活小鼠胸腺T淋巴细胞增殖过程中c-myc与核骨架蛋白的结合.实验结果显示,c-myc与核骨架蛋白的结合具有特异性,在淋巴细胞激活过程中c-myc与P₃₄/P₃₆核骨架蛋白及核纤层蛋白结合,并发生动态变化.     

Efficiencies of different genes and different tree-building methods in recovering a known vertebrate phylogeny

The relative efficiencies of different protein-coding genes of the mitochondrial genome and different tree-building methods in recovering a known vertebrate phylogeny (two whale species, cow, rat, mouse, opossum, chicken, frog, and three bony fish species) was evaluated. The tree-building methods examined were the neighbor joining (NJ), minimum evolution (ME), maximum parsimony (MP), and maximum likelihood (ML), and both nucleotide sequences and deduced amino acid sequences were analyzed. Generally speaking, amino acid sequences were better than nucleotide sequences in obtaining the true tree (topology) or trees close to the true tree. However, when only first and second codon positions data were used, nucleotide sequences produced reasonably good trees. Among the 13 genes examined, Nd5 produced the true tree in all tree-building methods or algorithms for both amino acid and nucleotide sequence data. Genes Cytb and Nd4 also produced the correct tree in most tree-building algorithms when amino acid sequence data were used. By contrast, Co2, Nd1, and Nd41 showed a poor performance. In general, large genes produced better results, and when the entire set of genes was used, all tree-building methods generated the true tree. In each tree-building method, several distance measures or algorithms were used, but all these distance measures or algorithms produced essentially the same results. The ME method, in which many different topologies are examined, was no better than the NJ method, which generates a single final tree. Similarly, an ML method, in which many topologies are examined, was no better than the ML star decomposition algorithm that generates a single final tree. In ML the best substitution model chosen by using the Akaike information criterion produced no better results than simpler substitution models. These results question the utility of the currently used optimization principles in phylogenetic construction. Relatively simple methods such as the NJ and ML star decomposition algorithms seem to produce as good results as those obtained by more sophisticated methods. The efficiencies of the NJ, ME, MP, and ML methods in obtaining the correct tree were nearly the same when amino acid sequence data were used. The most important factor in constructing reliable phylogenetic trees seems to be the number of amino acids or nucleotides used.      

Purification and characterization of the rat liver vasopressin (V1) receptor

Utilizing a proteoliposome reconstitution system, we have purified the rat liver V1 vasopressin receptor to near homogeneity. The receptor was purified approximately 21,000-fold from rat liver membranes, using differential detergent solubilization, size exclusion gel filtration, lectin affinity, and ion-exchange chromatography. The purified receptor exhibits a Kd of 6 nM, when, prior to solubilization, the membranes were exposed to 1 microM vasopressin. This resulted in the association of a pertussis toxin-insensitive guanine nucleotide-binding protein with the receptor during most of the purification procedure. In the absence of this association, the receptor had a Kd of approximately 30 nM. Association of the receptor with a G-protein was confirmed by the ability of vasopressin to stimulate the hydrolysis of [gamma-32P]GTP. The specific activity of the vasopressin-stimulated hydrolysis was 25 nmol/min/mg, approximately 8,000-fold higher than values obtained with crude reconstituted receptor preparations. Cross-linking of 125I-vasopressin to a partially purified preparation of receptor demonstrated that the receptor had a molecular weight of approximately 68,000 under reducing conditions, and 58,000 under nonreducing conditions. The purification procedure may prove useful in purifying a number of small peptide hormone receptors (e.g. bradykinin, angiotensin II) and perhaps their associated G-proteins as well.