A novel R-stereoselective amidase from Pseudomonas sp. MCI3434 acting on piperazine-2-tert-butylcarboxamide.

A novel amidase acting on (R,S)-piperazine-2-tert-butylcarboxamide was purified from Pseudomonas sp. MCI3434 and characterized. The enzyme acted R-stereoselectively on (R,S)-piperazine-2-tert-butylcarboxamide to yield (R)-piperazine-2-carboxylic acid, and was tentatively named R-amidase. The N-terminal amino acid sequence of the enzyme showed high sequence identity with that deduced from a gene named PA3598 encoding a hypothetical hydrolase in Pseudomonas aeruginosa PAO1. The gene encoding R-amidase was cloned from the genomic DNA of Pseudomonas sp. MCI3434 and sequenced. Analysis of 1332 bp of the genomic DNA revealed the presence of one open reading frame (ramA) which encodes the R-amidase. This enzyme, RamA, is composed of 274 amino acid residues (molecular mass, 30 128 Da), and the deduced amino acid sequence exhibits homology to a carbon-nitrogen hydrolase protein (PP3846) from Pseudomonas putida strain KT2440 (72.6% identity) and PA3598 protein from P. aeruginosa strain PAO1 (65.6% identity) and may be classified into a new subfamily in the carbon-nitrogen hydrolase family consisting of aliphatic amidase, beta-ureidopropionase, carbamylase, nitrilase, and so on. The amount of R-amidase in the supernatant of the sonicated cell-free extract of an Escherichia coli transformant overexpressing the ramA gene was about 30 000 times higher than that of Pseudomonas sp. MCI3434. The intact cells of the E. coli transformant could be used for the R-stereoselective hydrolysis of racemic piperazine-2-tert-butylcarboxamide. The recombinant enzyme was purified to electrophoretic homogeneity from cell-free extract of the E. coli transformant overexpressing the ramA gene. On gel-filtration chromatography, the enzyme appeared to be a monomer. It had maximal activity at 45 degrees C and pH 8.0, and was completely inactivated in the presence of p-chloromercuribenzoate, N-ethylmaleimide, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Ag+, Cd2+, Hg2+, or Pb2+. RamA had hydrolyzing activity toward the carboxamide compounds, in which amino or imino group is connected to beta- or gamma-carbon, such as beta-alaninamide, (R)-piperazine-2-carboxamide (R)-piperidine-3-carboxamide, D-glutaminamide and (R)-piperazine-2-tert-butylcarboxamide. The enzyme, however, did not act on the other amide substrates for the aliphatic amidase despite its sequence similarity to RamA.     

Characterization of EMU,the Arabidopsis homolog of the yeast THO complex member HPR1

Diverse and precise control is essential for eukaryotic gene expression. This is accomplished through the recruitment of a myriad of proteins to a nascent messenger RNA (mRNA) to mediate modifications, such as capping, splicing, 3′-end processing, and export. Despite being important for every cell, however, the mechanism by which the formation of diverse messenger ribonucleoprotein (mRNP) particles contributes to maintaining intricate systems in the multicellular organism remains incompletely defined. We identified and characterized a mutant gene named erecta mRNA under-expressed (emu) that leads to the defective mRNA accumulation of ERECTA, a developmental regulator in the model plant Arabidopsis thaliana. EMU encodes a protein homologous to a component of the THO complex that is required for the generation of functional mRNPs. Further analysis suggested that EMU is genetically associated with SERRATE, HYPONASTIC LEAVES1, and ARGONAUTE1, which are required for proper RNA maturation or action. Furthermore, mutations in another THO-related gene led to embryonic lethality. These findings support the presence and importance of the THO-related complex in plants as well as yeast and vertebrates.     

l-Stereoselective amino acid amidase with broad substrate specificity from Brevundimonas diminuta: characterization of a new member of the leucine aminopeptidase family

Brevundimonas diminuta TPU 5720 produces an amidase acting l-stereoselectively on phenylalaninamide. The enzyme (LaaA_Bd) was purified to electrophoretic homogeneity by ammonium sulfate fractionation and four steps of column chromatography. The final preparation gave a single band on SDS-PAGE with a molecular weight of ≈53,000. The native molecular weight of the enzyme was about 288,000 based on gel filtration chromatography, suggesting that the enzyme is active as a homohexamer. It had maximal activity at 50°C and pH 7.5. LaaA_Bd lost its activity almost completely on dialysis against potassium phosphate buffer (pH 7.0), and the amidase activity was largely restored by the addition of Co²⁺ ions. The enzyme was, however, inactivated in the presence of ethylenediaminetetraacetic acid even in the presence of Co²⁺, suggesting that LaaA_Bd is a Co²⁺-dependent enzyme. LaaA_Bd had hydrolyzing activity toward a broad range of l-amino acid amides including l-phenylalaninamide, l-glutaminamide, l-leucinamide, l-methioninamide, l-argininamide, and l-2-aminobutyric acid amide. Using information on the N-terminal amino acid sequence of the enzyme, the gene encoding LaaA_Bd was cloned from the chromosomal DNA of the strain and sequenced. Analysis of 4,446 bp of the cloned DNA revealed the presence of seven open-reading frames (ORFs), one of which (laaA _Bd ) encodes the amidase. LaaA_Bd is composed of 491 amino acid residues (calculated molecular weight 51,127), and the deduced amino acid sequence exhibits significant similarity to that of ORFs encoding hypothetical cytosol aminopeptidases found in the genomes of Caulobacter crescentus, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Mesorhizobium loti, and Agrobacterium tumefaciens, and leucine aminopeptidases, PepA, from Rickettsia prowazekii, Pseudomonas putida ATCC 12633, and Escherichia coli K-12. The laaA _Bd gene modified in the nucleotide sequence upstream from its start codon was overexpressed in an E. coli transformant. The activity of the recombinant LaaA_Bd in cell-free extracts of the E. coli transformant was 25.9 units mg⁻¹ with l-phenylalaninamide as substrate, which was 50 times higher than that of B. diminuta TPU 5720.     

Characteristic effect of an anticancer dinuclear platinum(II) complex on the higher-order structure of DNA

It is known that a 1,2,3-triazolato-bridged dinuclear platinum(II) complex, [{cis-Pt(NH₃)₂}₂(μ-OH)(μ-1,2,3-ta-N ¹,N ²)](NO₃)₂ (AMTA), shows high in vitro cytotoxicity against several human tumor cell lines and circumvents cross-resistance to cisplatin. In the present study, we examined a dose- and time-dependent effect of AMTA on the higher-order structure of a large DNA, T4 phage DNA (166 kbp), by adapting single-molecule observation with fluorescence microscopy. It was found that AMTA induces the shrinking of DNA into a compact state with a much higher potency than cisplatin. From a quantitative analysis of the Brownian motion of individual DNA molecules in solution, it became clear that the density of a DNA segment in the compact state is about 2,000 times greater than that in the absence of AMTA. Circular dichroism spectra suggested that AMTA causes a transition from the B to the C form in the secondary structure of DNA, which is characterized by fast and slow processes. Electrophoretic measurements indicated that the binding of AMTA to supercoiled DNA induces unwinding of the double helix. Our results indicate that AMTA acts on DNA through both electrostatic interaction and coordination binding; the former causes a fast change in the secondary structure from the B to the C form, whereas the latter promotes shrinking in the higher-order structure as a relatively slow kinetic process. The shrinking effect of AMTA on DNA is attributable to the possible increase in the number of bridges along a DNA molecule. It is concluded that AMTA interacts with DNA in a manner markedly different from that of cisplatin.     

TRANSIENT LOCALIZATION OF γ-TUBULIN AROUND THE CENTRIOLES IN THE NUCLEAR DIVISION OF BOERGESENIA FORBESII (SIPHONOCLADALES, CHLOROPHYTA)

Mitosis in Boergesenia forbesii (Harvey) Feldman was studied by immunofluorescence microscopy using anti-β–tubulin, anti-γ–tubulin, and anti-centrin antibodies. In the interphase nucleus, one, two, or rarely three anti-centrin staining spots were located around the nucleus, indicating the existence of centrioles. Microtubules (MTs) elongated randomly from the circumference of the nuclear envelope, but distinct microtubule organizing centers could not be observed. In prophase, MTs located around the interphase nuclei became fragmented and eventually disappeared. Instead, numerous MTs elongated along the nuclear envelope from the discrete anti-centrin staining spots. Anti-centrin staining spots duplicated and migrated to the two mitotic poles. γ–Tubulin was not detected at the centrioles during interphase but began to localize there from prophase onward. The mitotic spindle in B. forbesii was a typical closed type, the nuclear envelope remaining intact during nuclear division. From late prophase, accompanying the chromosome condensation, spindle MTs could be observed within the nuclear envelope. A bipolar mitotic spindle was formed at metaphase, when the most intense staining of γ-tubulin around the centrioles could also be seen. Both spindle MT poles were formed inside the nuclear envelope, independent of the position of the centrioles outside. In early anaphase, MTs between separating daughter chromosomes were not detected. Afterward, characteristic interzonal spindle MTs developed and separated both sets of the daughter chromosomes. From late anaphase to telophase, γ-tubulin could not be detected around the centrioles and MT radiation from the centrioles became diminished at both poles. γ-Tubulin was not detected at the ends of the interzonal spindle fibers. When MTs were depolymerized with amiprophos methyl during mitosis, γ-tubulin localization around the centrioles was clearly confirmed. Moreover, an influx of tubulin molecules into the nucleus for the mitotic spindle occurred at chromosome condensation in mitosis.     

Three new asymmetric trans-amine(azole)dichloridoplatinum complexes that overcome cisplatin resistance and their reactions with 5′-GMP

Three new asymmetric platinum(II) complexes comprising an isopropylamine ligand trans to an azole ligand were synthesized and fully characterized by ¹H NMR, ¹⁹⁵Pt NMR, IR and elemental analysis. In addition the X-ray crystal structure of all three complexes was determined. The reaction kinetics of the complexes with DNA model base guanosine-5′-monophosphate (GMP) was studied, revealing reaction kinetics comparable to cisplatin. To gain insight in the complexes as potential antitumor agents, cytotoxicity assays were performed on a variety of human tumor cell lines. These assays showed the complexes all to possess cytotoxicity profiles comparable to cisplatin. Furthermore, the complexes largely retain their activity in a human ovarian carcinoma cell line resistant to cisplatin, A2780R, compared to the cisplatin sensitive parent cell line A2780. These results are of fundamental importance, illustrating how platinum complexes of trans geometry can show improved activity compared to cisplatin in both cisplatin sensitive and cisplatin resistant cell lines.     

Phenotypic Characterization of the Komeda Miniature Rat Ishikawa, an Animal Model of Dwarfism Caused by a Mutation in Prkg2

The Komeda miniature rat Ishikawa (KMI) is a spontaneous animal model of dwarfism caused by a mutation in Prkg2, which encodes cGMP-dependent protein kinase type II (cGKII). This strain has been maintained as a segregating inbred strain for the mutated allele mri. In this study, we characterized the phenotype of the KMI strain, particularly growth traits, craniofacial measurements, and organ weights. The homozygous mutant (mri/mri) animals were approximately 70% to 80% of the size of normal, heterozygous (mri/+) animals in regard to body length, weight, and naso-occipital length of the calvarium, and the retroperitoneal fat of mri/mri rats was reduced greatly. In addition, among progeny of the (BN×KMI-mri/mri)F1×KMI-mri/mri backcross, animals with the KMI phenotype (mri/mri) were easily distinguished from those showing the wild-type phenotype (mri/+) by using growth traits such as body length and weight. Genetic analysis revealed that all of the backcrossed progeny exhibiting the KMI phenotype were homozygous for the KMI allele in the 1.2-cM region between D14Rat5 and D14Rat80 on chromosome 14, suggesting strongly that mri acts in a completely recessive manner. The KMI strain is the first and only rat model with a confirmed mutation in Prkg2 and is a valuable model for studying dwarfism and longitudinal growth traits in humans and for functional studies of cGKII.Abbreviations: cGKII, cGMP-dependent protein kinase type II; CNP, C-type natriuretic peptide; KMI, Komeda miniature rat IshikawaDwarfism is caused by both endocrinologic and nonendocrinologic defects. Most instances of dwarfism, including normal variants, are nonendocrinologic, and subjects retain growth hormone secretion. Although spontaneous rodent models of dwarfism with confirmed mutations have been reported—Snell dwarf mice with Pou1f1 (Pit1) mutation,¹⁴ Ames dwarf mice with Prop1 mutation,²² little mice with Ghrhr mutation,¹⁵ pygmy mice (also known as mini-mice) with Hmga2 (HMGI-C) mutation,²⁶ spontaneous dwarf rats with Gh mutation,²³ and rdw rats with Tg mutation^9,11—most of these are models of endocrinologic dwarfism. A few models of nonendocrinologic dwarfism have been produced by gene manipulation techniques, such as transgenic and knockout strategies, and include Col2a1-transgenic mice,^7,24 Col10a1-transgenic mice,¹⁰ and Fgfr3-knock-in mice.¹³A novel spontaneous dwarf mutation, miniature rat Ishikawa (mri), was discovered in a closed colony of Wistar rats at Ishikawa Animal Laboratory (Saitama, Japan) and has been maintained on the genetic background of Wistar rats. This mutant strain, previously termed Miniature Rat Ishikawa (MRI), has recently been established as a segregating inbred strain on the Wistar genetic background, designated Komeda Miniature rat Ishikawa (KMI). The breeding record suggested that the mutation was inherited in an autosomal recessive mode. KMI rats show no abnormality in the basal amounts or distribution of several hormones, including growth hormone, luteinizing hormone, follicle-stimulating hormone, prolactin, thyroid-stimulating hormone, and adrenocorticotropic hormone, but growth hormone response to growth hormone releasing hormone is decreased.²¹Using positional candidate cloning of mri, we recently identified a deletion mutation in Prkg2, which encodes cGMP-dependent protein kinase type II (cGKII), and clarified a role of cGKII as a molecular switch that couples cessation of proliferation and the start of hypertrophic differentiation of chondrocytes.² Longitudinal skeletal growth is achieved by endochondral ossification in the growth plate, in which chondrocyte hypertrophic differentiation is an important step. Due to the impaired coupling of proliferation and hypertrophic differentiation in the growth plate chondrocytes, homozygous mutant (mri/mri) animals show longitudinal growth retardation.In this study, we further characterize the phenotype of the KMI strain, including body length, body weight, organ weight, and craniofacial measurements. Furthermore, we describe phenotypic characteristics of the progeny produced from the (BN×KMI-mri/mri)F1×KMI-mri/mri backcross and provide updated genetic, physical, and comparative maps of the mri region.