Cloning and developmental expression of MARK/Par-1/MELK-related protein kinase xMAK-V in<Emphasis Type="Italic"> Xenopus laevis</Emphasis>

MAK-V/Hunk is a recently identified MARK/Par-1-related mammalian protein kinase. Although the precise function of this protein kinase is yet to be established, available data suggest its involvement in animals development and in the physiology of the nervous system. Here we report characterization of a cDNA encoding Xenopus laevis orthologue of MAK-V/Hunk protein kinase, xMAK-V. The in silico analysis also revealed MAK-V/Hunk orthologues in the fish Fugu rubripes and primitive chordate Ciona intestinalis but not in invertebrate species such as Drosophila melanogaster and Caenorhabditis elegans, suggesting that MAK-V/Hunk is a chordate-specific protein kinase. The expression of xmak-v in X. laevis embryos was analyzed using whole-mount in situ hybridization. Expression of xmak-v has been detected in all developmental stages studied including maternal expression in unfertilized eggs. The xmak-v mRNA has a predominant occurrence on the animal hemisphere of the egg, and this pattern of expression is sustained throughout cleavage and blastula stages. At the gastrula stage xmak-v expression is restricted to the ectoderm. In the later stage embryos xmak-v is expressed over the entire embryonic surface including the open neural plate at stage 15 and also in neural tube at stage 22. At tadpole stage xmak-v expression is strong in embryonic epidermis, nervous system and sensory organs, and is also obvious in perisomitic mesoderm and brachial arches.Edited by N. Satoh     

Transient outward current carried by inwardly rectifying K+ channels in guinea pig ventricular myocytes dialyzed with low-K+ solution

There have been periodic reports of nonclassic (4-aminopyridine insensitive) transient outward K+ current in guinea pig ventricular myocytes, with the most recent one describing a novel voltage-gated inwardly rectifying type. In the present study, we have investigated a transient outward current that overlaps inward Ca2+ current (I(Ca,L)) in myocytes dialyzed with 10 mM K+ solution and superfused with Tyrode's solution. Although depolarizations from holding potential (Vhp) -40 to 0 mV elicited relatively small inward I(Ca,L) in these myocytes, removal of external K+ or addition of 0.2 mM Ba2+ more than doubled the amplitude of the current. The basis of the enhancement of I(Ca,L) was the suppression of a large transient outward K+ current. Similar enhancement was observed when Vhp was moved to -80 mV and test depolarizations were preceded by short prepulses to -40 mV. Investigation of the time and voltage properties of the outward K+ transient indicated that it was inwardly rectifying and unlikely to be carried by voltage-gated channels. The outward transient was attenuated in myocytes dialyzed with high-Mg2+ solution, accelerated in myocytes dialyzed with 100 microM spermine solution, and abolished with time in myocytes dialyzed with ATP-free solution. These and other findings suggest that the outward transient is a component of classic "time-independent" inwardly rectifying K+ current.     

Immunotherapy with autologous tumor cells engineered to secrete Tag7/PGRP, an innate immunity recognition molecule

BACKGROUND: Recent studies indicate that the innate component of immune defense plays an important role in the establishment of antigen-specific immune response. We have previously isolated a novel mouse gene tag7/PGRP that was shown to be involved in the innate component of the immune system, and its insect homologue is an upstream mediator of Toll signaling in Drosophila. METHODS: Transiently or stably genetically modified mouse tumor cell lines expressing Tag7 were used. Tumor growth rate and animal survival were analyzed. Possible effector cells involved in tumor suppression were detected immunohistochemically. RESULTS: Transfection of mammary gland adenocarcinoma cells with the tag7 cDNA did not alter their growth rate in vitro but diminished their tumorogenicity in vivo in syngeneic and immunodeficient animals. Increased incidence of apoptosis was registered in the modified tumors. Transient expression of Tag7 by mouse melanoma M3 cells elicited protective immunity against parental tumor cells. Immunohistochemical analysis revealed that tumors after immunization with the genetically modified cells were infiltrated with Mac1(+) cells, B220(+) cells, and NK cells. Using nude mice we observed rejection of modified cells, but did not detect memory formation. CONCLUSIONS: We can conclude that secretion of the Tag7 protein by genetically modified cells can induce mobilization of antigen-presenting cells and innate effectors. Memory mechanisms are mediated by T cell response. For the first time our results demonstrate that local secretion of Tag7-the molecule involved in innate immunity-may play an important role in the induction of effective antitumor response in mice.     

Synthesis, spectral properties, and antitumor activity of a new axially substituted phthalocyanine complex of zirconium(IV) with citric acid

The new axially substituted phthalocyanine (pc) complex of zirconium(IV) with citric acid is reported. It has been shown that the replacement of two Cl-atoms with two citric acid fragments takes place as the result of the reaction between [ZrCl2(pc)] and citric acid. The complex [Zr(citrate)2(pc)] was formed. The spectroscopic properties of the synthesized compound in DMSO, RPMI 1640 medium with and without fetal calf serum (FCS), H2O, and buffer (Tris) solutions have been described. Antitumor activity of this compound has been studied. The cytostatic activity was observed in the concentration range of 6.1-9.0x10(9) molecules [Zr(citrate)2(pc)]/cell and occurred in 4-6 h after treatment with [Zr(citrate)2(pc)] solution.     

Similar active sites in lysostaphins and D-Ala-D-Ala metallopeptidases

Specific peptidases exist for nearly every amide linkage in peptidoglycan. In several cases, families of peptidoglycan hydrolases with different specificities turned out to be related. Here we show that lysostaphin-type peptidases and D-Ala-D-Ala metallopeptidases have similar active sites and share a core folding motif in otherwise highly divergent folds. The central Zn(2+) is tetrahedrally coordinated by two histidines, an aspartate, and a water molecule. The Zn(2+) chelating residues occur in the order histidine, aspartate, histidine in all sequences and contact the metal via the Nepsilon, the Odelta, and the Ndelta, respectively. The identity of the other active-site residues varies, but in all enzymes of known structure except for VanX, a conserved histidine is present two residues upstream of the second histidine ligand to the Zn(2+). As the same arrangement of active-site residues is also found in the N-terminal, cryptic peptidase domain of sonic hedgehog, we propose that this arrangement of active-site residues be called the "LAS" arrangement, because it is present in lysostaphin-type enzymes, D-Ala-D-Ala metallopeptidases, and in the cryptic peptidase in the N-domain of sonic hedgehog.     

Quantitative analysis of microtubule transport in growing nerve processes

In neurons, tubulin is synthesized primarily in the cell body, whereas the molecular machinery for neurite extension and elaboration of microtubule (MT) array is localized to the growth cone region. This unique functional and biochemical compartmentalization of neuronal cells requires transport mechanisms for the delivery of newly synthesized tubulin and other cytoplasmic components from the cell body to the growing axon. According to the polymer transport model, tubulin is transported along the axon as a polymer. Because the majority of axonal MTs are stationary at any given moment, it has been assumed that only a small fraction of MTs translocates along the axon by saltatory movement reminiscent of the fast axonal transport. Such intermittent "stop and go" MT transport has been difficult to detect or to exclude by using direct video microscopy methods. In this study, we measured the translocation of MT plus ends in the axonal shaft by expressing GFP-EB1 in Xenopus embryo neurons in culture. Formal quantitative analysis of MT assembly/disassembly indicated that none of the MTs in the axonal shaft were rapidly transported. Our results suggest that transport of axonal MTs is not required for delivery of newly synthesized tubulin to the growing nerve processes.     

Possible role of region 152-156 in the structural duality of a peptide fragment from sheep prion protein

The conformational conversion of the nonpathogenic "cellular" prion isoform into a pathogenic "scrapie" protease-resistant isoform is a fundamental event in the onset of transmissible spongiform encephalopathies (TSE). During this pathogenic conversion, helix H1 and its two flanking loops of the normal prion protein are thought to undergo a conformational transition into a beta-like structure. A peptide spanning helix H1 and beta-strand S2 (residues 142-166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. This peptide in aqueous solution, in contrast to many prion fragments studied earlier (1) is highly soluble and (2) does not aggregate until the millimolar concentration range, and (3) exhibits an intrinsic propensity to a beta-hairpin-like conformation at neutral pH. We found that this peptide can also fold into a helix H1 conformation when dissolved in a TFE/PB mixture. The structures of the peptide calculated by MD showed solvent-dependent internal stabilizing forces of the structures and evidenced a higher mobility of the residues following the end of helix H1. These data suggest that the molecular rearrangement of this peptide in region 152-156, particularly in position 155, could be associated with the pathogenic conversion of the prion protein.     

A novel structure of DNA repair protein RecO from Deinococcus radiodurans

Recovery of arrested replication requires coordinated action of DNA repair, replication, and recombination machineries. Bacterial RecO protein is a member of RecF recombination repair pathway important for replication recovery. RecO possesses two distinct activities in vitro, closely resembling those of eukaryotic protein Rad52: DNA annealing and RecA-mediated DNA recombination. Here we present the crystal structure of the RecO protein from the extremely radiation resistant bacteria Deinococcus radiodurans (DrRecO) and characterize its DNA binding and strand annealing properties. The RecO structure is totally different from the Rad52 structure. DrRecO is comprised of three structural domains: an N-terminal domain which adopts an OB-fold, a novel alpha-helical domain, and an unusual zinc-binding domain. Sequence alignments suggest that the multidomain architecture is conserved between RecO proteins from other bacterial species and is suitable to elucidate sites of protein-protein and DNA-protein interactions necessary for RecO functions during the replication recovery and DNA repair.     

Importance of the reverse Hoogsteen base pair 54-58 for tRNA function

To elucidate the general constraints imposed on the structure of the D- and T-loops in functional tRNAs, active suppressor tRNAs were selected in vivo from a combinatorial tRNA gene library in which several nucleotide positions of these loops were randomized. Analysis of the nucleotide sequences of the selected clones demonstrates that among the randomized nucleotides, the most conservative are nucleotides 54 and 58 in the T-loop. In most cases, they make the combination U54-A58, which allows the formation of the normal reverse Hoogsteen base pair. Surprisingly, other clones have either the combination G54-A58 or G54-G58. However, molecular modeling shows that these purine–purine base pairs can very closely mimic the reverse Hoogsteen base pair U-A and thus can replace it in the T-loop of a functional tRNA. This places the reverse Hoogsteen base pair 54-58 as one of the most important structural aspects of tRNA functionality. We suggest that the major role of this base pair is to preserve the conformation of dinucleotide 59–60 and, through this, to maintain the general architecture of the tRNA L-form.