The structure, biochemical properties, and immunogenicity of neurofilament peripheral regions are determined by phosphorylation state

Treatment of freshly isolated, bovine neurofilaments with Escherichia coli alkaline phosphatase removes over 90% of the phosphate groups from serine residues of the Mr 200,000 and 150,000 polypeptide components (NF200 and NF150). Dephosphorylated NF200 and NF150 remain associated with filaments, but migrate in sodium dodecyl sulfate gels with reduced apparent molecular weights. Unusual migration appears to be due to modification at regions of these polypeptides that are peripheral to the neurofilament backbone as defined by limited chymotryptic digestion. Over 90 monoclonal antibodies recognizing epitopes located within the peripheral domain of native NF200 all show reduced affinity for dephosphorylated NF200. A single monoclonal antibody binds within the filament-associated domain of NF200 and its recognition of NF200 is unaffected upon treatment of neurofilaments with phosphatase. Around 50% of our monoclonal antibodies that bind NF150 monospecifically and at epitopes within its peripheral domain have reduced affinities for NF150 from phosphatase-treated filaments, while the remaining 50% bind native and dephosphorylated NF150 equally well. The smallest neurofilament component (NF70) contains few phosphate groups, most of which remain after treatment of neurofilaments with phosphatase. The resulting form of NF70 migrates normally in gels and its recognition by antibodies is unchanged. We conclude that phosphorylation modifies the structure of the two larger neurofilament polypeptides along domains that are peripheral to the filamentous backbone and that these effects are more pronounced for NF200 than for NF150.     

Mitogenomic analysis of Montipora cactus and Anacropora matthai (cnidaria; scleractinia; acroporidae) indicates an unequal rate of mitochondrial evolution among Acroporidae corals

The complete nucleotide sequence of the mitochondrial (mt) genome was determined for specimens of the coral species Montipora cactus (Bernard 1897) and Anacropora matthai (Pillai 1973), representing two morphologically distinct genera of the family Acroporidae. These sequences were compared with the published mt genome sequence for the confamilial species, Acropora tenuis (Dana 1846). The size of the mt genome was 17,887 bp and 17,888 bp for M. cactus and A. matthai. Gene content and organization was found to be very similar among the three Acroporidae mt genomes with a group I intron occurring in the NADH dehyrogenase 5 (nad5) gene. The intergenic regions were also similar in length among the three corals. The control region located between the small ribosomal RNA (ms) and the cytochrome oxidase 3 (cox3) gene was significantly smaller in M. cactus and A. matthai (both 627 bp) than in A. tenuis (1086 bp). Only one set of repeated sequences was identified at the 3′-end of the control regions in M. cactus and A. matthai. A lack of the abundant repetitive elements which have been reported for A. tenuis, accounts for the relatively short control regions in M. cactus and A. matthai. Pairwise distances and relative rate analyses of 13 protein coding genes, the group I intron and the largest intergenic region, igr3, revealed significant differences in the rate of molecular evolution of the mt genome among the three species, with an extremely slow rate being seen between Montipora and Anacropora. It is concluded that rapid mt genome evolution is taking place in genus Acropora relative to the confamilial genera Montipora and Anacropora although all are within the relatively slow range thought to be typical of Anthozoa.     

The structure and organization of the human heavy neurofilament subunit (NF-H) and the gene encoding it.

Genomic clones for the largest human neurofilament protein (NF-H) were isolated, the intron/exon boundaries mapped and the entire protein-coding regions (exons) sequenced. The predicted protein contains a central region that obeys the structural criteria identified for alpha-helical 'rod' domains typically present in all IF protein components: it is approximately 310 amino acids long, shares amino acid sequence homology with other IF protein rod domains and displays the characteristic heptad repeats of apolar amino acids which facilitate coiled-coil interaction. Nevertheless, anomalies are noted in the structure of the NF-H rod which could explain observations of its poor homopolymeric assembly in vitro. The protein segment on the carboxy-terminal side of the human NF-H rod is uniquely long (greater than 600 amino acids) compared to other IF proteins and is highly charged (greater than 24% Glu, greater than 25% Lys), rich in proline (greater than 12%) and impoverished in cysteine, methionine and aromatic amino acids. Its most remarkable feature is a repetitive sequence that covers more than half its length and includes the sequence motif, Lys-Ser-Pro (KSP) greater than 40 times. Together with the recent identification of the serine in KSP as the main target for NF-directed protein kinases in vivo, this repetitive character explains the massive phosphorylation of the NF-H subunit that can occur in axons. The human NF-H gene has three introns, two of which interrupt the protein-coding sequence at identical points to introns in the genes for the two smaller NF proteins, NF-M and NF-L.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurofilaments from ox spinal nerves. Isolation, disassembly, reassembly and cross-linking properties.

An isolation procedure for neurofilaments from ox spinal nerves is described where the triplet polypeptides (which have molecular weights of 205 000, 158 000 and 72 000) constitute more than 80% of the preparation. Soon after purification, the neurofilaments form a gel that is stable for many weeks. The purified neurofilaments disassemble in low-salt buffers at pH greater than 7.0 into soluble particles that contain all of the triplet polypeptides. Greater than 90% of the protein can reassemble to form filaments. The thiol-containing residues in the filaments can be cross-linked. Analyses of the complexes formed show that in the filament the 205 000-mol.wt. components are arranged to that they can be cross-linked to themselves and to the 158 000-mol.wt. polypeptides, and that the 72 000-mol.wt. components are arranged so that their thiol groups can be cross-linked together.     

Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.      

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Subunits of the eukaryotic cytosolic chaperonin CCT do not always behave as components of a uniform hetero-oligomeric particle

The chaperonin CCT is an hetero-oligomeric molecular chaperone complex. Studies in yeast suggest each of its eight gene products are required for its major identified functions in producing native tubulins and actins. However, it is unclear whether these eight components always form a single particle, covering all functions, or else can also exist as heterogeneous mixtures and/or free subunits in cells. Using mouse P19 embryonal carcinoma cells, which divide rapidly, yet in retinoic acid adopt a neuronal phenotype, admixed with occasional (approximately 10%) fibroblast-like cells, together with a panel of peptide-specific antibodies raised to 7 of the 8 CCT subunits we show that; (1) adoption of a post mitotic phenotype is accompanied by reduced CCT protein expression, significantly more so for CCTbeta, CCTdelta, CCTepsilon, and CCTtheta than for CCTalpha (TCP-1), CCTgamma and CCTzeta; (2) CCTalpha is detected preferentially over other subunits in neurites of P19 neurons; (3) small amounts of CCTalpha and gamma are localised in nuclei (i.e. are not exclusively cytoplasmic), selectively so compared with other subunits; (4) numerous cytosolic foci exist in the cytoplasm which, when detected by double immunofluorescence can contain only one of the subunits probed for; (5) while a "core" chaperonin particle can be immunoprecipitated under native conditions, epitope access is modified both by nucleotides and by non-CCT co-precipitating proteins. Collectively, these findings indicate that CCT subunits are not only components of the hetero-oligomeric chaperonin particle but exist as significant populations of free subunits or smaller oligomers in cells.     

Polynitroxyl-albumin (PNA) plus tempol attenuate lung capillary leak elicited by prolonged intestinal ischemia and reperfusion(1)

Stable nitroxyl radicals (nitroxides) are potential antioxidant drugs, and we have previously reported that linking nitroxide to biological macromolecules can improve therapeutic activity in at least two ways. First, polynitroxylated compounds such as polynitroxyl human serum albumin (PNA) are a novel class of high molecular weight, extracellular antioxidants. Second, compounds such as PNA can prolong the half-life of free (unbound, low molecular weight) nitroxides such as 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl (Tempol) in vivo. Unlike PNA, Tempol can readily access the intracellular compartment. Thus PNA can act alone in the extracellular compartment, or in concert with Tempol, to provide additional antioxidant protection within cells. In this study, we compared the abilities of PNA, Tempol, and the combination of PNA + Tempol to prevent lung microvascular injury secondary to prolonged gut ischemia (I, 120 min) and reperfusion (R, 20 min) in the rat. Pulmonary capillary filtration coefficient (K(f,c)) and lung neutrophil retention (tissue myeloperoxidase activity, MPO) were measured in normal, isolated rat lungs perfused with blood harvested from I/R rats. Blood donor rats were treated with drug during ischemia. Gut I/R resulted in a marked increase in pulmonary capillary coefficient and lung MPO. PNA + Tempol, but not PNA alone or Tempol alone, at the doses used, prevented the development of lung leak. None of the treatments had an effect on lung neutrophil retention. Anti-inflammatory therapeutic activity appeared to correlate with blood Tempol level: in the presence of PNA, blood Tempol levels were maintained in the 50-100 microM range vs. essentially undetectable levels shortly after Tempol was administered alone. In this model of lung injury secondary to prolonged gut I/R, lung capillary leak was prevented when the membrane-permeable compound Tempol was maintained in its active, free radical state by PNA.