Desulforubrerythrin from <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>, a novel multidomain protein

A novel multidomain metalloprotein from Campylobacter jejuni was overexpressed in Escherichia coli, purified, and extensively characterized. This protein is isolated as a homotetramer of 24-kDa monomers. According to the amino acid sequence, each monomer was predicted to contain three structural domains: an N-terminal desulforedoxin-like domain, followed by a four-helix bundle domain harboring a non-sulfur μ-oxo diiron center, and a rubredoxin-like domain at the C-terminus. The three predicted iron sites were shown to be present and were studied by a combination of UV–vis, EPR, and resonance Raman spectroscopies, which allowed the determination of the electronic and redox properties of each site. The protein contains two FeCys₄ centers with reduction potentials of +240 mV (desulforedoxin-like center) and +185 mV (rubredoxin-like center). These centers are in the high-spin configuration in the as-isolated ferric form. The protein further accommodates a μ-oxo-bridged diiron site with reduction potentials of +270 and +235 mV for the two sequential redox transitions. The protein is rapidly reoxidized by hydrogen peroxide and has a significant NADH-linked hydrogen peroxide reductase activity of 1.8 μmol H₂O₂ min⁻¹ mg⁻¹. Owing to its building blocks and its homology to the rubrerythrin family, the protein is named desulforubrerythrin. It represents a novel example of the large diversity of the organization of domains exhibited by this enzyme family.     

Reconstruction of tubular structures in three-dimensional collagen gel culture using proximal tubular epithelial cells voided in human urine

Human proximal tubular (PT) epithelial cells were isolated from urine and monoclonally cultured as monolayers for 1 wk, after which they were subcultured between two layers of collagen gel, designated a "collagen gel sandwich." Under these culture conditions, PT cells formed three-dimensional tubular structures exhibiting distinct polarized cell morphology. Scanning and transmission electron microscopic studies showed that they bore numerous microvilli at the apical surface and that they closely contacted the collagen gel at the basal surface. These studies indicate that PT cells exfoliated in urine still exhibit the potential to proliferate and form organized structures mimicking in vivo tubules. Because of the current lack of useful culture systems for human tubular epithelial cells originating from kidney tissue, we suggest that this unique culture system using voided PT cells in urine could open up new avenues to study not only the mechanisms of morphogenesis but also the physiology of human PT cells.     

Co-expression of soybean glycinins A1aB1b and A3B4 enhances their accumulation levels in transgenic rice seed

The soybean major storage protein glycinin is encoded by five genes, which are divided into two subfamilies. Expression of A3B4 glycinin in transgenic rice seed reached about 1.5% of total seed protein, even if expressed under the control of strong endosperm-specific promoters. In contrast, expression of A1aB1b glycinin reached about 4% of total seed protein. Co-expression of the two proteins doubled accumulation levels of both A1aB1b and A3B4 glycinins. This increase can be largely accounted for by their aggregation with rice glutelins, self-assembly and inter-glycinin interactions, resulting in the enrichment of globulin and glutelin fractions and a concomitant reduction of the prolamin fraction. Immunoelectron microscopy indicated that the synthesized A1aB1b glycinin was predominantly deposited in protein body-II (PB-II) storage vacuoles, whereas A3B4 glycinin is targeted to both PB-II and endoplasmic reticulum (ER)-derived protein body-I (PB-I) storage structures. Co-expression with A1aB1b facilitated targeting of A3B4 glycinin into PB-II by sequestration with A1aB1b, resulting in an increase in the accumulation of A3B4 glycinin.     

Cloning and Characterization of Two Novel Human cDNAs (NELL1 and NELL2) Encoding Proteins with Six EGF-like Repeats

From a human fetal-brain cDNA library we isolated two novel genes encoding peptides containing six EGF-like repeats. Both showed significant homologies with nel, a gene strongly expressed in neural tissues of chicken. The cDNAs, designated NELL1 (nel-like, type 1) and NELL2 (nel-like, type 2), contained open reading frames encoding 810 and 816 amino acids, respectively. NELL2 is strongly expressed in brain of adult and fetus but only weakly in fetal kidney. NELL1 and NELL2 were mapped by FISH to chromosomal bands 11p15.1–p15.2 and 12q13.11–q13.12, respectively.     

Myosin-II reorganization during mitosis is controlled temporally by its dephosphorylation and spatially by Mid1 in fission yeast

Cytokinesis in many eukaryotes requires an actomyosin contractile ring. Here, we show that in fission yeast the myosin-II heavy chain Myo2 initially accumulates at the division site via its COOH-terminal 134 amino acids independently of F-actin. The COOH-terminal region can access to the division site at early G2, whereas intact Myo2 does so at early mitosis. Ser1444 in the Myo2 COOH-terminal region is a phosphorylation site that is dephosphorylated during early mitosis. Myo2 S1444A prematurely accumulates at the future division site and promotes formation of an F-actin ring even during interphase. The accumulation of Myo2 requires the anillin homologue Mid1 that functions in proper ring placement. Myo2 interacts with Mid1 in cell lysates, and this interaction is inhibited by an S1444D mutation in Myo2. Our results suggest that dephosphorylation of Myo2 liberates the COOH-terminal region from an intramolecular inhibition. Subsequently, dephosphorylated Myo2 is anchored by Mid1 at the medial cortex and promotes the ring assembly in cooperation with F-actin.     

Participation of hup gene product in ori2-dependent replication of fertility plasmid F

The fertility plasmid F'gal was not stably maintained in a hupA-hupB double mutant of Escherichia coli. Moreover, mini-F plasmids pFZY1, pFTC1 and pFTC2 were unable to transform the double mutant, though these plasmids efficiently transformed cells harboring a hupA or hupB single mutation. The composite plasmid pFHS1, which consists of the f5 DNA fragment of F plasmid and the whole DNA of a pSC101 derivative that carries a temperature-sensitive mutation for DNA replication, was not stably maintained in the hup double mutant at 42°C. These findings strongly suggest that HU protein is required for ori2-dependent replication of the F plasmid.     

Dissection of genotype-phenotype associations in rice grains using metabolome quantitative trait loci analysis

A comprehensive and large‐scale metabolome quantitative trait loci (mQTL) analysis was performed to investigate the genetic backgrounds associated with metabolic phenotypes in rice grains. The metabolome dataset consisted of 759 metabolite signals obtained from the grains of 85 lines of rice (Oryza sativa, Sasanishiki × Habataki back‐crossed inbred lines). Metabolome analysis was performed using four mass spectrometry pipelines to enhance detection of different classes of metabolites. This mQTL analysis of a wide range of metabolites highlighted an uneven distribution of 802 mQTLs on the rice genome, as well as different modes of metabolic trait (m‐trait) control among various types of metabolites. The levels of most metabolites within rice grains were highly sensitive to environmental factors, but only weakly associated with mQTLs. Coordinated control was observed for several groups of metabolites, such as amino acids linked to the mQTL hotspot on chromosome 3. For flavonoids, m‐trait variation among the experimental lines was tightly governed by genetic factors that alter the glycosylation of flavones. Many loci affecting levels of metabolites were detected by QTL analysis, and plausible gene candidates were evaluated by in silico analysis. Several mQTLs profoundly influenced metabolite levels, providing insight into the control of rice metabolism. The genomic region and genes potentially responsible for the biosynthesis of apigenin‐6,8‐di‐C‐α‐l‐ arabinoside are presented as an example of a critical mQTL identified by the analysis.     

On the hoplonemertean Sagaminemertes nagaiensis (Iwata, 1957), with consideration of its systematics

Summary The polystiliferous hoplonemertean Sagaminemertes nagaiensis (Iwata, 1957) has been redescribed.A distinct pre-cerebral region is wanting. The rhynchocoel opens directly at the tip of the head, whilst the mouth opens seperately below the ventral commissure of the brain which is situated at the anterior end of the head. The dorsal ganglia are well developed and extend posteriorly far behind the ventral ganglia. The cerebral sense organs are situated behind the brain and possess posterior ciliated canals. Cephalic glands are poorly represented, but ocelli are well developed. The sickle-shaped basis of the proboscis bears about 20 central stylets. The caecal appendages of the rhynchocoel are arranged pseudometamerically throughout the body. The foregut is straight, consisting of mouth, oesophagus, stomach and pylorus. Intestinal caeca and lateral diverticula are present. The cephalic lacunae have no anterior anastomosis. A cerebral lacuna is present, there is a rhynchocoel vessel, but transverse vessels are absent. The protonephridia extend throughout the foregut region. Mature spermatogonia are well developed.A systematic discussion of Sagaminemertes nagaiensis suggests that it is taxonomically close to Siboganemertes weberi in the archireptantic family Siboganemertidae.