High efficiency penetration of antibody-immobilized nanoneedle thorough plasma membrane for in situ detection of cytoskeletal proteins in living cells.

BACKGROUND: The field of structural dynamics of cytoskeletons in living cells is gathering wide interest, since better understanding of cytoskeleton intracellular organization will provide us with not only insights into basic cell biology but may also enable development of new strategies in regenerative medicine and cancer therapy, fields in which cytoskeleton-dependent dynamics play a pivotal role. The nanoneedle technology is a powerful tool allowing for intracellular investigations, as it can be directly inserted into live cells by penetrating through the plasma membrane causing minimal damage to cells, under the precise manipulation using atomic force microscope. Modifications of the nanoneedles using antibodies have allowed for accurate mechanical detection of various cytoskeletal components, including actin, microtubules and intermediate filaments. However, successful penetration of the nanoneedle through the plasma membrane has been shown to vary greatly between different cell types and conditions. In an effort to overcome this problem and improve the success rate of nanoneedle insertion into the live cells, we have focused here on the fluidity of the membrane lipid bilayer, which may hinder nanoneedle penetration into the cytosolic environment. RESULTS: We aimed to reduce apparent fluidity of the membrane by either increasing the approach velocity or reducing experimental temperatures. Although changes in approach velocity did not have much effect, lowering the temperature was found to greatly improve the detection of unbinding forces, suggesting that alteration in the plasma membrane fluidity led to increase in nanoneedle penetration. CONCLUSIONS: Operation at a lower temperature of 4 °C greatly improved the success rate of nanoneedle insertion to live cells at an optimized approach velocity, while it did not affect the binding of antibodies immobilized on the nanoneedle to vimentins for mechanical detection. As these experimental parameters can be applied to various cell types, these results may improve the versatility of the nanoneedle technology to other cell lines and platforms.

The pattern of GPI-80 expression is a useful marker for unusual myeloid maturation in peripheral blood.

Myeloid-derived suppressor cells (MDSCs) have a wide spectrum of immunosuppressive activity; control of these cells is a new target for improving clinical outcomes in cancer patients. MDSCs originate from unusual differentiation of neutrophils or monocytes induced by inflammatory cytokines, including granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage (GM)-CSF. However, MDSCs are difficult to detect in neutrophil or monocyte populations because they are not uniform cells, resembling both neutrophils and monocytes; thus, they exist in a heterogeneous population. In this study, we investigated GPI-80, a known regulator of Mac-1 (CD11b/CD18) and associated closely with neutrophil maturation, to clarify this unusual differentiation. First, we demonstrated that the mean fluorescence intensity (MFI) of GPI-80 and coefficient of variation (CV) of GPI-80 were increased by treatment with G-CSF and GM-CSF, respectively, using a human promyelocytic leukaemia (HL60) cell differentiation model. To confirm the value of GPI-80 as a marker of unusual differentiation, we measured GPI-80 expression and MDSC functions using peripheral blood cells from metastatic renal cell carcinoma patients. The GPI-80 CV was augmented significantly in the CD16hi neutrophil cell population, and GPI-80 MFI was increased significantly in the CD33hi monocyte cell population. Furthermore, the GPI-80 CV in the CD16hi population was correlated inversely with the proliferative ability of T cells and the GPI-80 MFI of the CD33hi population was correlated with reactive oxygen species production. These results led us to propose that the pattern of GPI-80 expression in these populations is a simple and useful marker for unusual differentiation, which is related to MDSC functions.

Differential localization of non-muscle myosin II isoforms and phosphorylated regulatory light chains in human MRC-5 fibroblasts.

We investigated the localization of non-muscle myosin II isoforms and mono- (at serine 19) and diphosphorylated (at serine 19 and threonine 18) regulatory light chains (RLCs) in motile and non-motile MRC-5 fibroblasts. In migrating cells, myosin IIA localized to the lamella and throughout the posterior region. Myosin IIB colocalized with myosin IIA to the posterior region except at the very end. Diphosphorylated RLCs were detected in the restricted region where myosin IIA was enriched. In non-motile cells, myosin IIA was enriched in peripheral stress fibers with diphosphorylated RLCs, but myosin IIB was not. Our results suggest that myosin IIA may be highly activated by diphosphorylation of RLCs and primarily involved in cell migration.

Novel findings regarding photoinduced commitments of G1-, S- and G2-phase cells to cell-cycle transitions in darkness and dark-induced G1-, S- and G2-phase arrests in Euglena.

Effects of light and darkness on cell-cycle progression were studied in the log-linear photoautotrophic growth mode of Euglena gracilis. We found that there are light-dependent restriction points in the post-G1 phases, quite in contrast to Chlamydomonas, where a light-dependent restriction is known to exist only in the G1 phase. Thus, in E. gracilis, there are photoinduced commitments of G1-, S- and G2-phase cells that allow them to progress to the G1, S and G2 phases in darkness, and there are dark-induced G1-, S- and G2-phase arrests. In darkness, only committed cells were able to progress to the committed phases (G1, S or G2), whereas uncommitted cells were unable to undergo a cell-cycle transition. Whether or not cells were induced to commit by irradiation, they were eventually arrested somewhere in the G1, S or G2 (but not M) phase within 14 h of being transferred to darkness. We also describe the dependence of photoinduced commitment on light intensity and discuss the results as they relate to cell-cycle progression in continuous light.

Export of Thermus thermophilus alkaline phosphatase via the twin-arginine translocation pathway in Escherichia coli.

The bacterial twin-arginine translocation (Tat) pathway is distinct from the Sec system by its remarkable capacity to export folded enzymes. To address the question whether the two systems are capable of translocating homologous enzymes catalyzing the same reaction, we cloned the tap gene encoding Thermus thermophilus alkaline phosphatase (Tap) and expressed it in Escherichia coli. Unlike the alkaline phosphatase of E. coli, which is translocated through the Sec system and then activated in the periplasm, Tap was exported exclusively via the Tat pathway and active Tap precursor was observed in the cytoplasm. These results demonstrate that two sequence and functional related enzymes are exported by distinct protein transport systems, which may play an integral role in the bacterial adaptation to their environment during the evolution.

Analysis of the effect of accumulation of amino acid replacements on activity of 3-isopropylmalate dehydrogenase from Thermus thermophilus.

A newly selected cold-adapted mutant 3-isopropylmalate dehydrogenase (IPMDH) from a random mutant library was a double mutant containing the mutations I11V and S92F that were found in cold-adapted mutant IPMDHs previously isolated. To elucidate the effect of each mutation on enzymatic activity, I11V and six multiple mutant IPMDHs were constructed and analyzed. All of the multiple mutant IPMDHs were found to be improved in catalytic activity at moderate temperatures by increasing the k(cat) with a simultaneous increase of K(m) for the coenzyme NAD(+). k(cat) was improved by a decrease in the activation enthalpy, DeltaH( not equal). The multiple mutants did not show large reduction in thermal stability, and one of them showed enhanced thermal stability. Mutation from I11 to V was revealed to have a stabilizing effect. Mutants showed increased thermal stability when the mutation I11V was combined. This indicates that it is possible to construct mutants with enhanced thermal stability by combining stabilizing mutation. No additivity was observed for the thermodynamic properties of catalytic reaction in the multiple mutant IPMDHs, implying that the structural changes induced by the mutations were interacting with each other. This indicates that careful and detailed tuning is required for enhancing activity in contrast to thermal stability.

Complete genome sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain7.

The complete genomic sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain7 which optimally grows at 80 degrees C, at low pH, and under aerobic conditions, has been determined by the whole genome shotgun method with slight modifications. The genomic size was 2,694,756 bp long and the G + C content was 32.8%. The following RNA-coding genes were identified: a single 16S-23S rRNA cluster, one 5S rRNA gene and 46 tRNA genes (including 24 intron-containing tRNA genes). The repetitive sequences identified were SR-type repetitive sequences, long dispersed-type repetitive sequences and Tn-like repetitive elements. The genome contained 2826 potential protein-coding regions (open reading frames, ORFs). By similarity search against public databases, 911 (32.2%) ORFs were related to functional assigned genes, 921 (32.6%) were related to conserved ORFs of unknown function, 145 (5.1%) contained some motifs, and remaining 849 (30.0%) did not show any significant similarity to the registered sequences. The ORFs with functional assignments included the candidate genes involved in sulfide metabolism, the TCA cycle and the respiratory chain. Sequence comparison provided evidence suggesting the integration of plasmid, rearrangement of genomic structure, and duplication of genomic regions that may be responsible for the larger genomic size of the S. tokodaii strain7 genome. The genome contained eukaryote-type genes which were not identified in other archaea and lacked the CCA sequence in the tRNA genes. The result suggests that this strain is closer to eukaryotes among the archaea strains so far sequenced. The data presented in this paper are also available on the internet homepage (http://www.bio.nite.go.jp/E-home/genome_list-e.html/).

Highly efficient transfection system for functional gene analysis in adult amphibian lens regeneration.

The analysis of newt lens regeneration has been an important subject in developmental biology. Recently, it has been reported that the genes involved in the normal eye development are also expressed in the regenerative process of lens regeneration in the adult newt. However, functional analysis of these genes has not been possible, because there is no system to introduce genes efficiently into the cells involved in the regeneration. In the present study, lipofection was used as the method for gene transfer in cultured pigmented iris cells that can transdifferentiate into lens cells in newt lens regeneration. Positive expression of a reporter gene was obtained in more than 70% of cells. In addition, the aggregate derived from gene-transfected cells maintained its expression at a high level for a long time within the host tissue. To verify the effectiveness of this model system with a reporter gene in lens regeneration, Pax6, which is suggested to be involved in normal eye development and lens regeneration, was transfected. Ectopic expression of lens-specific crystallins was obtained in cells that show no such activity in normal lens regeneration. These results made it possible for the first time to analyze the molecular mechanism of lens regeneration in the adult newt.

Ancestral residues stabilizing 3-isopropylmalate dehydrogenase of an extreme thermophile: experimental evidence supporting the thermophilic common ancestor hypothesis.

Ancestral amino acid residues were inferred for 3-isopropylmalate dehydrogenase (IPMDH), and were introduced into the enzyme of an extreme thermophile, Sulfolobus sp. strain 7. The thermostability of the mutant enzymes was compared with that of the wild type enzyme. At least five of the seven mutants tested showed higher thermal stability than the wild type IPMDH. The results are compatible with the hyperthermophilic universal ancestor hypothesis. The results also provide a new method for designing thermostable enzymes. The method only relies on the first dimensional structures of homologous enzymes that can be obtained from genetic databases.

Selection of stabilized 3-isopropylmalate dehydrogenase of Saccharomyces cerevisiae using the host-vector system of an extreme thermophile, Thermus thermophilus.

A leuB strain of Thermus thermophilus TTY1, was transformed with a plasmid vector that directed expression of 3-isopropylmalate dehydrogenase (IPMDH) of Saccharomyces cerevisiae encoded by the LEU2 gene. The original strain could not grow at 50 degrees C without leucine, probably because of the low stability of S. cerevisiae IPMDH. The mutants that could grow without leucine were selected at 50 degrees, 60 degrees, 62 degrees, 65 degrees, 67 degrees, and 70 degrees C, step by step. All the mutant strains except for one isolated at 50 degrees C accumulated mutations. Mutations were serially accumulated: Glu255Val, Asn43Tyr, Ala62Thr, Asn110Lys, and Alal 12Val, respectively, at each step. The analyses of residual activity after heat treatment and the denaturation profile as monitored by circular dichroism showed that thermal stability was increased with accumulation of the mutations. The kinetic parameters of most mutant enzymes were similar to those of the wild type. However, some mutant enzymes showed a reverse correlation between stability and activity: the enzymes with a large increase in thermal stability showed lower activity. Although the wild-type enzyme is unstable in the absence of glycerol, the stabilizing effect of glycerol was not observed for all the mutant enzymes containing the Glu255Val substitution, which is assumed to be located at the hydrophobic interface between two subunits.

Crystal structures of mutants of Thermus thermophilus IPMDH adapted to low temperatures.

Random mutagenesis on thermophilic 3-isopropylmalate dehydrogenases (IPMDH; EC 1.1.1.85) produced mutant enzymes which adapt to low temperatures. These mutants had higher activity at lower temperatures than the wild-type enzyme without losing high thermostability. Here we report three structures of the mutants of Thermus thermophilus IPMDH determined by X-ray diffraction which was adapted to a low-temperature environment. Two of them have unstable coenzyme binding states and the other one probably has a stable substrate binding state. The present research suggests that the adaptation is correlated with the binding of either coenzyme or the substrate.

Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures.

Random mutagenesis coupled with screening of the active enzyme at a low temperature was applied to isolate cold-adapted mutants of a thermophilic enzyme. Four mutant enzymes with enhanced specific activities (up to 4.1-fold at 40 degrees C) at a moderate temperature were isolated from randomly mutated Thermus thermophilus 3-isopropylmalate dehydrogenase. Kinetic analysis revealed two types of cold-adapted mutants, i.e. k(cat)-improved and K(m)-improved types. The k(cat)-improved mutants showed less temperature-dependent catalytic properties, resulting in improvement of k(cat) (up to 7.5-fold at 40 degrees C) at lower temperatures with increased K(m) values mainly for NAD. The K(m)-improved enzyme showed higher affinities toward the substrate and the coenzyme without significant change in k(cat) at the temperatures investigated (30-70 degrees C). In k(cat)-improved mutants, replacement of a residue was found near the binding pocket for the adenine portion of NAD. Two of the mutants retained thermal stability indistinguishable from the wild-type enzyme. Extreme thermal stability of the thermophilic enzyme is not necessarily decreased to improve the catalytic function at lower temperatures. The present strategy provides a powerful tool for obtaining active mutant enzymes at lower temperatures. The results also indicate that it is possible to obtain cold-adapted mutant enzymes with high thermal stability.

Occurrence of D-Amino Acids and a pyridoxal 5'-phosphate-dependent aspartate racemase in the acidothermophilic archaeon, Thermoplasma acidophilum.

Free D-amino acid content in some archaea was investigated and D-forms of several amino acids were found in them. In the acidothermophilic archaeon, Thermoplasma acidophilum, the proportion of D-aspartate (D-Asp) to total Asp was as high as 39.7%. Crude extracts of Thermoplasma acidophilum had Asp-specific racemase activity that was pyridoxal 5'-phosphate (PLP)-dependent. The relative insensitivity to a SH-modifying reagent distinguished this activity from those of the PLP-independent Asp racemases found in other hyperthermophilic archaea (Matsumoto, M., et al., J. Bacteriol. 181, 6560-6563 1999). Thus, high levels of d-Asp should be produced by a new type(s) of Asp-specific racemase in Thermoplasma acidophilum, although the function of d-Asp in this archaeon remains unknown.

Cold adaptation of the thermophilic enzyme 3-isopropylmalate dehydrogenase.

We have performed random mutagenesis coupled with selection to isolate mutant enzymes with high catalytic activities at low temperature from thermophilic 3-isopropylmalate dehydrogenase (IPMDH) originally isolated from Thermus thermophilus. Five cold-adapted mutant IPMDHs with single-amino-acid substitutions were obtained and analyzed. Kinetic analysis revealed that there are two types of cold-adapted mutant IPMDH: k(cat)-improved (improved in k(cat)) and K(m)-improved (improved in k(cat)/K(m)) types. To determine the mechanisms of cold adaptation of these mutants, thermodynamic parameters were estimated and compared with those of the Escherichia coli wild-type IPMDH. The Delta G(m) values for Michaelis intermediate formation of the k(cat)-improved-type enzymes were larger than that of the T. thermophilus wild-type IPMDH and similar to that of the E. coli wild-type IPMDH. The Delta G(m) values of K(m)-improved-type enzymes were smaller than that of the T. thermophilus wild-type IPMDH. Fitting of NAD(+) binding was improved in the K(m)-improved-type enzymes. The two types of cold-adapted mutants employed one of the two strategies of E. coli wild-type IPMDH: relative destabilization of the Michaelis complex in k(cat)-improved-type, and destabilization of the rate-limiting step in K(m)-improved type mutants. Some cold-adapted mutant IPMDHs retained thermostability similar to that of the T. thermophilus wild-type IPMDH.

Quinone profiles of Thermoplasma acidophilum HO-62.

Quinones of Thermoplasma acidophilum HO-62 were analyzed by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance. Menaquinone, methionaquinone, and 2-trans and 2-cis forms of thermoplasmaquinone were identified. The relative amount of thermoplasmaquinone increased under anaerobic conditions, and those of menaquinone and methionaquinone increased under aerobic conditions.

High thermal stability of 3-isopropylmalate dehydrogenase from Thermus thermophilus resulting from low DeltaC(p) of unfolding.

To characterize the thermal stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, urea-induced unfolding of the enzyme and of its mesophilic counterpart from Escherichia coli was investigated at various temperatures. The unfolding curves were analyzed with a three-state model for E.coli IPMDH and with a two-state model for T.thermophilus IPMDH, to obtain the free energy change DeltaG degrees of each unfolding process. Other thermodynamic parameters, enthalpy change DeltaH, entropy change DeltaS and heat capacity change DeltaC(p), were derived from the temperature dependence of DeltaG degrees. The main feature of the thermophilic enzyme was its lower dependence of DeltaG degrees on temperature resulting from a low DeltaC(p). The thermophilic IPMDH had a significantly lower DeltaC(p), 1.73 kcal/mol.K, than that of E.coli IPMDH (20.7 kcal/mol.K). The low DeltaC(p) of T.thermophilus IPMDH could not be predicted from its change in solvent-accessible surface area DeltaASA. The results suggested that there is a large structural difference between the unfolded state of T.thermophilus and that of E.coli IPMDH. Another responsible factor for the higher thermal stability of T.thermophilus IPMDH was the increase in the most stable temperature T(s). The DeltaG degrees maximum of T.thermophilus IPMDH was much smaller than that of E.coli IPMDH. The present results clearly demonstrated that a higher melting temperature T(m) is not necessarily accompanied by a higher DeltaG degrees maximum.

Functional characterization of vertebrate nonmuscle myosin IIB isoforms using Dictyostelium chimeric myosin II.

The alternatively spliced isoform of nonmuscle myosin II heavy chain B (MHC-IIB) with an insert of 21 amino acids in the actin-binding surface loop (loop 2), MHC-IIB(B2), is expressed specifically in the central nervous system of vertebrates. To examine the role of the B2 insert in the motor activity of the myosin II molecule, we expressed chimeric myosin heavy chain molecules using the Dictyostelium myosin II heavy chain as the backbone. We replaced the Dictyostelium native loop 2 with either the noninserted form of loop 2 from human MHC-IIB or the B2-inserted form of loop 2 from human MHC-IIB(B2). The transformant Dictyostelium cells expressing only the B2-inserted chimeric myosin formed unusual fruiting bodies. We then assessed the function of chimeric proteins, using an in vitro motility assay and by measuring ATPase activities and binding to F-actin. We demonstrate that the insertion of the B2 sequence reduces the motor activity of Dictyostelium myosin II, with reduction of the maximal actin-activated ATPase activity and a decrease in the affinity for actin. In addition, we demonstrate that the native loop 2 sequence of Dictyostelium myosin II is required for the regulation of the actin-activated ATPase activity by phosphorylation of the regulatory light chain.

Glycogen storage disease type Ib without neutropenia.

We report 2 patients with atypical glycogen storage disease type Ib without neutropenia or infectious complications. Neither patient was deficient in hepatic glucose-6-phosphatase activities in microsome-disrupted homogenates; both had mutations in the glucose-6-phosphate transporter gene, suggesting an allelic variant of glycogen storage disease type Ib.

B2 exon splicing of nonmuscle myosin heavy chain IIB is differently regulated in developing and adult rat brain.

Two isoforms of nonmuscle myosin heavy chain IIB (MHC-IIB) are generated by alternative splicing; MHC-IIB(B2) differs from MHC-IIB(DeltaB2) by the insertion of B2 exon cassette near the actin binding region. Here we examined expressions of the two splice variants in developing and adult rat brains by in situ hybridization with isoform-specific oligonucleotide probes. In adult, MHC-IIB(DeltaB2) mRNA was highly expressed in neurons of the cerebral cortex, hippocampus, and cerebellum, whereas MHC-IIB(B2) mRNA was mainly distributed in the brainstem and cerebellum, with the highest level in Purkinje cells. During development, MHC-IIB(DeltaB2) mRNA was predominantly expressed in various regions of embryonic and neonatal brains, whereas MHC-IIB(B2) mRNA was low during embryonic stages. Up-regulation of MHC-IIB(B2) started in the cerebellum during early postnatal stages when dendritogenesis and synaptogenesis occur actively in Purkinje cells. We further employed immunofluorescence using two antibodies (one recognizing both splicing variants and another specific to MHC-IIB(B2)), and found similar and dense localization in cell bodies and dendrites of Purkinje cells. Therefore, splicing of the B2 exon cassette undergoes distinct temporal and spatial regulations in the brain in vivo, and the different exon usage seems unlikely to affect the somato-dendritic localization of MHC-IIB.

The mouse N-acetylgalactosamine-6-sulfate sulfatase (Galns) gene: cDNA isolation, genomic characterization, chromosomal assignment and analysis of the 5'-flanking region.

Deficiency of lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS) leads to mucopolysaccharidosis IV A (MPS IV A), for which there is no definitive treatment so far. Although a number of mutations of the GALNS gene of MPS IV A patients have been described, pathogenesis of the disorder still remains elusive. In order to facilitate in vivo studies using model animals for MPS IV A, we isolated and performed molecular characterization of the mouse homolog of human GALNS. The 2.3-kb cDNA contains a 1560-bp open reading frame encoding 520 amino acid residues. The coding region has 84% similarity to the human GALNS cDNA at amino acid level. The mouse Galns gene was mapped by interspecific backcross analysis to the distal region of chromosome 8 where it co-segregates with Aprt. Northern blot analysis showed a wide expression of a single-copy gene, being higher especially in liver and kidney. The Galns gene was isolated from S129vJ genomic library and its genomic organization was characterized. The mouse Galns gene was about 50-kb long and organized into 14 exons and 13 introns. All intron-exon splice junctions conformed to the GT/AG consensus sequence except exon 8/intron 8 junction. Primer extension shows multiple transcription initiation sites between -44 and -75 although major transcription initiation site was observed at -90 bp from the ATG codon. The 5'-flanking region lacks canonical TATA and CAAT box sequences, but is G+C rich with 10 GC boxes (potential Sp1 binding sites), characteristic of a housekeeping gene promoter.