Calcium Influx into Human Neuroblastoma Cells Induces ALZ-50 Immunoreactivity: Involvement of Calpain-Mediated Hydrolysis of Protein Kinase C

Abstract: Calcium influx into SH-SY5Y human neuroblastoma cells after ionophore treatment or transient permeabilization in calcium-containing medium increased ALZ-50 immunoreactivity markedly. This increase was prevented by inhibitors active against calpain or against protein kinase C (PKC), suggesting that both of these enzymes were required to mediate the effect of calcium influx on ALZ-50 immunoreactivity. Treatment with PKC activator TPA increased ALZ-50 immunoreactivity in the absence of calcium influx or after intracellular delivery of the specific calpain inhibitor calpastatin, indicating that the influence of PKC was downstream from that of calpain. Calcium influx also resulted in μ-calpain autolysis (one index of calpain activation) and the transient appearance of PKM (i.e., free PKC catalytic subunits, generated by calpain-mediated cleavage of the regulatory and catalytic PKC domains). Inhibition of calpain within intact cells resulted in a dramatic increase in steady-state levels of total τ (migrating at 46–52 kDa) but resulted in a relatively minor increase in 68-kDa ALZ-50-immunoreactive τ isoforms. Although calcium influx into intact cells resulted in accumulation of ALZ-50 immunoreactivity, total τ levels were, by contrast, rapidly depleted. Incubation of isolated fractions with calpain in the presence of calcium indicated that ALZ-50-immunoreactive τ isoforms were more resistant to calpain-mediated proteolysis than were non-ALZ-50 reactive τ isoforms. These data therefore indicate that calpain may regulate τ levels directly via proteolysis and indirectly through PKC activation. A consequence of the latter action is altered τ phosphorylation, perhaps involving one or more kinase cascades, and the preferential accumulation of ALZ-50-immunoreactive τ isoforms due to their relative resistance to degradation. These findings provide a basis for the possibility that disregulation of calcium homeostasis may contribute to the pathological levels of conversion of τ to A68 by hyperactivation of the calpain/PKC system.     

Interaction between yeast Cdc6 protein and B-type cyclin/Cdc28 kinases.

During purification of recombinant Cdc6 expressed in yeast, we found that Cdc6 interacts with the critical cell cycle, cyclin-dependent protein kinase Cdc28. Cdc6 and Cdc28 can be coimmunoprecipitated from extracts, Cdc6 is retained on the Cdc28-binding matrix p13-agarose, and Cdc28 is retained on an affinity column charged with bacterially produced Cdc6. Cdc6, which is a phosphoprotein in vivo, contains five Cdc28 consensus sites and is a substrate of the Cdc28 kinase in vitro. Cdc6 also inhibits Cdc28 histone H1 kinase activity. Strikingly, Cdc6 interacts preferentially with B-type cyclin/Cdc28 complexes and not Cln/Cdc28 in log-phase cells. However, Cdc6 does not associate with Cdc28 when cells are blocked at the restrictive temperature in a cdc34 mutant, a point in the cell cycle when the B-type cyclin/Cdc28 inhibitor p40Sic1 accumulates and purified p40Sic1 inhibits the Cdc6/Cdc28 interaction. Deletion of the Cdc28 interaction domain from Cdc6 yields a protein that cannot support growth. However, when overproduced, the mutant protein can support growth. Furthermore, whereas overproduction of wild-type Cdc6 leads to growth inhibition and bud hyperpolarization, overproduction of the mutant protein supports growth at normal rates with normal morphology. Thus, the interaction may have a role in the essential function of Cdc6 in initiation and in restraining mitosis until replication is complete.     

异育淇鲫及其亲本血清生化组成的比较研究

应用Beckman 42型临床生化分析仪测定异育淇鲫(淇鲫♀×兴国红鲤♂) 及其亲本血清的生化指标,包括总蛋白,白蛋白,尿素氮,血糖、乳酸脱氢酶,碱性磷酸酶,谷草转氨酶和羟丁酸脱氢酶,经t检验发现异育淇鲫与其母本的血清生化指标均无明显差异,而与其父本均有十分显著的差异。本文从代谢方面论证了淇鲫与兴国红鲤属间“杂交”,异源精子只起刺激作用,而并不产生生物学效应。结果与传统的雌核发育相吻合。     

大鼠胃泌酸腺细胞膜和肝匀浆对五肽胃泌素及其类似物的降解

 本文用HPLC分离五肽胃泌素(Pentagastrin,Boc-β-Ala-Trp-Met-Asp-Phe·NH_2,PG_(1-5))及其N-端无保护基的TFA·五肽(β-Ala-Trp-Met-Asp-Pne·NH_2·TFA,TFA·PA_(1-5))被胃泌酸细胞膜和肝匀浆降解之产物,并对它们的氨基酸组成作了分析,又用薄层层析作了进一步验证,从而提出了酶促降解时可能的酶切位点。实验结果表明,N-端的Boc-基团能大幅度增加对酶降解的抵抗力,这从一个方面解释了为什么商品五肽胃泌素的泌酸活性大于其它胃泌素类似物的原因。     

Triphenylmethylphosphonium cation distribution as a measure of hormone-induced alterations in white adipocyte membrane potential

Summary Triphenylmethylphosphonium (TPMP⁺) partitions into the mitochondrial and cytosolic compartments in the rat white adipocyte in a potential-dependent fashion. The relationship between [³H]TPMP⁺ distribution, intracellular cAMP generation and lipolysis in response to hormones and cAMP-mimetic compounds was examined. Half-maximal [³H]TPMP⁺ efflux and glycerol release were produced by 15 and 9nm adrenocorticotropin, 170 and 110nm 1-epinephrine, 70 and 27 m isobutylmethylxanthine and 800 and 750 m dibutyryl cAMP, respectively. Hormone-stimulated cAMP generation was also correlated with [³H]TPMP⁺ efflux and lipolysis in terms of concentration dependency. In kinetic experiments, glycerol release and [³H]TPMP⁺ efflux in response to adrenocorticotropin or cholera toxin proceeded over a similar time course, whereas an earlier rise in cAMP generation was detected.The depolarizing effect of lipolytic compounds was localized to the mitochondrial compartment. When cells were incubated in elevated-[K⁺] _c buffer, the stimulatory effect of dibutyryl cAMP on [³H]TPMP⁺ efflux and lipolysis persisted, suggesting that maintenance of the plasma membrane potential is not critical for demonstration of these responses.When the extracellular concentration of serum albumin, which provides binding sites for free fatty acids, was increased from 1 to 3% an increase in glycerol release and a decrease in [³H]TPMP⁺ efflux was observed. We suggest that intracellular free fatty acid accumulation in response to lipolytic agents causes dissipation of the mitochondrial membrane potential and efflux of [³H]TPMP⁺ from the organelle and cell.     

Gbu Glycine Betaine Porter and Carnitine Uptake in Osmotically Stressed Listeria monocytogenes Cells

The food-borne pathogen Listeria monocytogenes grows actively under high-salt conditions by accumulating compatible solutes such as glycine betaine and carnitine from the medium. We report here that the dominant transport system for glycine betaine uptake, the Gbu porter, may act as a secondary uptake system for carnitine, with a K_m of 4 mM for carnitine uptake and measurable uptake at carnitine concentrations as low as 10 μM. This porter has a K_m for glycine betaine uptake of about 6 μM. The dedicated carnitine porter, OpuC, has a K_m for carnitine uptake of 1 to 3 μM and a V_max of approximately 15 nmol/min/mg of protein. Mutants lacking either opuC or gbu were used to study the effects of four carnitine analogs on growth and uptake of osmolytes. In strain DP-L1044, which had OpuC and the two glycine betaine porters Gbu and BetL, triethylglycine was most effective in inhibiting growth in the presence of glycine betaine, but trigonelline was best at inhibiting growth in the presence of carnitine. Carnitine uptake through OpuC was inhibited by γ-butyrobetaine. Dimethylglycine inhibited both glycine betaine and carnitine uptake through the Gbu porter. Carnitine uptake through the Gbu porter was inhibited by triethylglycine. Glycine betaine uptake through the BetL porter was strongly inhibited by trigonelline and triethylglycine. These results suggest that it is possible to reduce the growth of L. monocytogenes under osmotically stressful conditions by inhibiting glycine betaine and carnitine uptake but that to do so, multiple uptake systems must be affected.     

Molecular and genetic characterization and physical mapping of 11 new markers detecting multiallele restriction fragment length polymorphisms on the short arm of human chromosome 3

Summary Genetic markers with high degrees of polymorphisms are of vital importance in the construction of high resolution (2–4 cM) linkage maps of human chromosomes as specified in the short-term goals of the Human Genome Initiative. In this paper, we report on molecular and genetic characterization and physical localization of 11 new multiallele restriction fragment length polymorphism markers on human chromosome 3p. Ten of these represent three- and four-allele polymorphisms of the base substitution type probably at two adjacent restriction sites. One has been identified as a novel minisatellite sequence comprising a variable copy number tandem repeat array of a G/T-rich 79-bp sequence. This collection of multiallele polymorphic (PIC values: 0.40–0.60) markers should prove valuable and increase the resolution power of the available chromosome 3p genetic markers.     

Proton NMR comparison of noncovalent and covalently cross-linked complexes of cytochrome c peroxidase with horse, tuna, and yeast ferricytochromes c.

Proton NMR spectroscopy at 500 and 361 MHz has been used to characterize the noncovalent or electrostatic complexes of yeast cytochrome c peroxidase (CcP) with horse, tuna, yeast isozyme-1, and yeast isozyme-2 ferricytochromes c and the covalently cross-linked complexes of cytochrome c peroxidase with horse and yeast isozyme-1 ferricytochromes c. Under the conditions employed in this work, the stoichiometry of the predominant complex formed in solution (which totaled greater than 90% of complex formed) was found to be 1:1 in all cases. These studies have elucidated significant differences in the proton NMR absorption spectra and the one-dimensional nuclear Overhauser effect difference spectra of the complexes, depending on the specific species of ferricytochrome c incorporated. In particular, the results indicate that the noncovalent complexes formed between CcP and physiological redox partners (yeast isozyme-1 or yeast isozyme-2 ferricytochromes c) are distinctly different from the noncovalent complexes formed between CcP and ferricytochromes c from horse and tuna. Parallel chemical cross-linking studies carried out using mixtures of cytochrome c peroxidase with horse ferricytochrome c, and cytochrome c peroxidase with yeast isozyme-1 ferricytochrome c further emphasize such cytochrome c-dependent differences, with only the covalently cross-linked complex of physiological redox partners (cytochrome c peroxidase/yeast isozyme-1) displaying NMR spectra characteristic of a heterogeneous mixture of different 1:1 complexes. Finally, one-dimensional nuclear Overhauser effect experiments have proven valuable in selectively and efficiently probing the protein-protein interface in these complexes, including the environment around the cytochrome c heme 3-methyl group and Phe-82.     

A DNA-activated protein kinase from HeLa cell nuclei.

A DNA-activated protein kinase (DNA-PK) was purified from nuclei of HeLa cells. Activity was associated with a single high-molecular-mass (approximately-300,000 Da) polypeptide when analyzed by gel filtration, denaturing polyacrylamide gel electrophoresis, and Western immunoblotting using a monoclonal antibody that also inhibits enzyme activity. Nuclear localization was indicated by subcellular fractionation and confirmed by immunofluorescence on whole cells. Double-stranded DNA stimulated phosphorylation of the 300-kDa polypeptide in purified preparations as well as phosphorylation of the exogenous substrates alpha-casein, simian virus 40 large T antigen, and the human heat shock protein hsp90. Autophosphorylation led to inactivation of the enzyme. The phosphorylation of casein was stimulated over 30-fold by DNA and was specific for serine and threonine residues. Bovine serum albumin and histone H1 were poor substrates for DNA-PK, and no phosphorylation of immunoglobulin G or histones other than H1 was observed. Supercoiled or heat-denatured DNA and synthetic double-stranded RNA or RNA-DNA copolymers did not stimulate casein phosphorylation by DNA-PK. Interaction of the enzyme with DNA in the absence of exogenous substrates was demonstrated by thermal inactivation and gel mobility shifts. These characteristics identify DNA-PK as distinct from other protein kinases described in the literature and suggest that activation by DNA is an important feature of the enzyme's in vivo function.