Silkworm midgut proteins interacting with a hemolymph protease inhibitor, CI-8

CI-8 is the chymotrypsin inhibitor in hemolymph from the silkworm, Bombyx mori. It occurs in the midgut at the spinning stage of larva, but little information on the mechanism of its uptake in the midgut is available. We found that two polypeptides interacting with CI-8 are in the midgut membrane, and we purified them using a biotinylated CI-8, viz., p29 and p60, having molecular sizes of 29 kDa and 60 kDa respectively. The structures of p29 and p60 were examined by N-terminal amino acid sequencing and peptide mass mapping, including tryptic digestion. p29 was highly similar to the matured 19G1-30K lipoprotein from hemolymph, but p60 was novel. Purified p29 was recognized by anti-19G1-30K antibody, and was confirmed to be similar to 19G1-30K. The antibody also neutralized the CI-8 binding ability of p29 in the midgut membrane. p29 and p60 are perhaps proteinaceous factors involved in the uptake of CI-8 into the midgut through the membrane.     

In vitro immunization can elicit the expansion of diverse repertoire of B cells from peripheral blood mononuclear cells

We previously developed an in vitro immunization (IVI) protocol of human peripheral blood mononuclear cells (PBMC) for generating antigen-specific human antibodies. In order to clarify whether IVI protocolinduces antigen-specific B cell responses in PBMC, we analyzed family gene usage and sequence of the variable region gene of immunoglobulin heavy chain (VH gene) of the antibody produced from the in vitro immunized PBMC. Sequence homology analyses of VH gene demonstrated that a larger repertoire of B cells can be sensitized with mite-extract than with cholera toxin B subunit and rice allergen. Further, antigen-specific B cells were efficiently expanded by using CpG oligodeoxynucleotide as adjuvant. These results suggest that appropriate combination of sensitizing antigen and adjuvant is primarily important for expansion of antigen-specific B cells in IVI protocol.     

Development of first photoresponsive prodrug of paclitaxel

A prodrug of paclitaxel which has a coumarin derivative conjugated to the amino acid moiety of isotaxel (O-acyl isoform of paclitaxel) has been synthesized. The prodrug was selectively converted to isotaxel by visible light irradiation (430 nm) with the cleavage of coumarin. Finally, paclitaxel was released by subsequent spontaneous O-N intramolecular acyl migration.     

Cold-Tolerant Crop Species Have Greater Temperature Homeostasis of Leaf Respiration and Photosynthesis Than Cold-Sensitive Species

Some plant species show constant rates of respiration and photosynthesismeasured at their respective growth temperatures (temperaturehomeostasis), whereas others do not. However, it is unclearwhat species show such temperature homeostasis and what factorsaffect the temperature homeostasis. To analyze the inherentability of plants to acclimate respiration and photosynthesisto different growth temperatures, we examined 11 herbace-ouscrops with different cold tolerance. Leaf respiration (R_area)and photosynthetic rate (P_area) under high light at 360 µll^–1 CO₂ concentrations were measured in plants grown at15 and 30°C. Cold-tolerant species showed a greater extentof temperature homeostasis of both R_area and P_area than cold-sensitivespecies. The underlying mechanisms which caused differencesin the extent of temperature homeostasis were examined. Theextent of temperature homeostasis of P_area was not determinedby differences in leaf mass and nitrogen content per leaf area,but by differences in photosynthetic nitrogen use efficiency(PNUE). Moreover, differences in PNUE were due to differencesin the maximum catalytic rate of Rubisco, Rubisco contents andamounts of nitrogen invested in Rubisco. These findings indicatedthat the temperature homeostasis of photosynthesis was regulatedby various parameters. On the other hand, the extent of temperaturehomeostasis of R_area was unrelated to the maximum activity ofthe respiratory enzyme (NAD-malic enzyme). The R_area/P_area ratiowas maintained irrespective of the growth temperatures in allthe species, suggesting that the extent of temperature homeostasisof R_area interacted with the photosynthetic rate and/or thehomeostasis of photosynthesis.     

Multiplex real-time PCR for the simultaneous detection of herpes simplex virus, human herpesvirus 6, and human herpesvirus 7

A simultaneous detection system to quantify HSV, HHV-6, and HHV-7 DNA via multiplex real-time PCR using different fluorochromes was developed. The minimum quantitative level established via this multiplex assay was four copies per reaction for HSV type 1, four copies for HHV-6, and three copies for HHV-7, respectively. The dynamic range encompassed at least six orders of magnitude. The system was specific and reproducible even in the presence of large amounts of other viral DNA. We then applied this multiplex real-time PCR assay to 105 CSF specimens obtained from subjects less than 15 years old in whom a diagnosis of viral encephalitis/encephalopathy was suspected on clinical grounds. The detection rate for each viral DNA was 6.7% for HSV, 9.5% for HHV-6, and 1.9% for HHV-7. These results indicate that our system is reliable and may be useful for the rapid diagnosis of viral encephalitis/encephalopathy.     

A Novel Endoplasmic Reticulum Export Signal: PROLINE AT THE +2-POSITION FROM THE SIGNAL PEPTIDE CLEAVAGE SITE*

NUCB1 (nucleobindin 1) is a Golgi-localized soluble protein with a signal peptide and multiple functional domains. We reported recently that NUCB1 is a negative regulator of the unfolded protein response that activates various endoplasmic reticulum (ER)-originating signaling pathways. In that report, we also showed that Golgi localization of NUCB1 was essential to regulate the unfolded protein response. However, the localization mechanism of NUCB1 is still unknown. Here, we report that the proline residue at the +2-position (Pro⁺²) from the signal peptide cleavage site is the determinant of NUCB1 protein export from the ER and subsequent transport to the Golgi. Fusion of the N-terminal amino acids 1–35 peptide region, including both signal peptide (amino acids 1–26) and Pro⁺², was sufficient for enhanced green fluorescent protein to localize in the Golgi, whereas single amino acid mutation of Pro⁺² resulted in defective export from the ER without affecting the protein maturation process. Furthermore, we demonstrated that Pro⁺² was important for the enhanced green fluorescent protein fusion protein to concentrate at a transport vesicle formation site within the ER, often termed the ER exit site. Interestingly, such a Pro⁺² has also been functionally conserved in other Golgi-localized soluble proteins, Cab45 (Ca²⁺-binding protein of 45 kDa), reticulocalbin 1, and calumenin. Our findings indicate that Pro⁺² can function as a novel ER export signal of some Golgi proteins.NUCB1 (nucleobindin 1), also known as calnuc, was first identified as a soluble secretory 55-kDa protein (461 amino acids) in lupus-prone mice with the lymphoproliferation (lpr) mutation (1). NUCB1 has also been shown to be secreted in culture supernatant of a murine B cell line established from the mice (2). Later studies also demonstrated that NUCB1 is expressed ubiquitously and localizes in the Golgi apparatus of intact cells (3, 4). NUCB1 contains multiple putative functional domains, including an N-terminal endoplasmic reticulum (ER)² signal peptide, a DNA binding site, a leucine zipper domain, two EF-hand Ca²⁺-binding sites, a nuclear localization signal, and G-protein-binding and cyclooxygenase-binding domains (1, 5, 6). Consistently, NUCB1 has been reported to function in various cellular processes, including osteogenesis, inflammation, autoimmunity, intracellular signaling, and cancer (6–10).Newly synthesized, premature NUCB1 protein is first targeted into the ER via its N-terminal ER signal peptide. After removal of the signal peptide in the ER, a mature NUCB1 protein is transported to the Golgi apparatus and then secreted to the extracellular matrix (11). NUCB1 in the Golgi pool is probably involved in establishing the agonist-mobilizable Golgi Ca²⁺ store (3). Furthermore, the Golgi-localized NUCB1 regulates the unfolded protein response, which is a cellular stress response that triggers various events, such as ER-resident molecular chaperone induction, translational repression, and apoptosis under ER stress conditions (12). On the other hand, extracellular NUCB1 has been suggested to serve as a modulator of matrix maturation in bone, based on the observations that NUCB1 is secreted by osteoblasts and osteocytes and can, indeed, be detected in the osteoid extracellular matrix (7, 13). Thus, Golgi transport and subsequent secretion of NUCB1 seem to be important to exert the protein''s activity, but little is known about its transport regulation mechanism.In eukaryotic cells, a tremendous variety of soluble and membrane cargo proteins are packaged into transport vesicles at the ER. Vesicle formation on the ER membrane begins with the assembly of a coat protein complex II (COPII) (14). This COPII coat consists of Sar1, Sec23-Sec24, and Sec13-Sec31 complexes that are sequentially recruited to the ER membrane. Sar1 is a small GTPase that regulates coat assembly and disassembly. To assemble the COPII coat, Sar1-GTP transiently associates with an export cargo protein and then binds to Sec23-Sec24, which in turn attracts Sec13-Sec31 (14). Polymerization of the formed COPII coat, which occurs at the so-called ER exit site (ERES), triggers transport vesicle budding on the ER membrane (14, 15). Then the vesicles fuse with the VTC compartment (vesiculo-tubular clusters, also called ERGIC) that mediates further protein transport to the Golgi apparatus. Cargo proteins are then carried to their final destinations, such as organelle, cell surface membrane, and extracellular matrix (14).Recent studies reveal that some transmembrane cargoes contain specific motifs to be selectively concentrated in the transport vesicle within the ER. This sorting motif is called the ER export signal. Representative ER export signals are the diacidic motif (DXE), dihydrophobic (LL) motif, and diaromatic motif (FF, YY) that have been found in vesicular stomatitis virus glycoprotein, ERGIC-53, and Emp46p, respectively (16–18). These export signals are present in the cytoplasmic region of the cargo proteins and mediate their interaction with the COPII complex at the outer side of the ER membrane, resulting in concentration in the newly formed budding vesicle. On the other hand, the soluble type of cargo proteins require their cargo receptor to be sorted into the COPII vesicle, because they cannot interact directly with COPII complex, since these proteins have no cytoplasmic region. Although recent studies have reported the existence of the cargo receptor and functional ER export signals found in some soluble cargo proteins, little is known about many other cargo receptors and the export signals of soluble cargo proteins (19, 20).Here, we report that Pro²⁸, which is located at the +2-position (Pro⁺²) from the signal peptide cleavage site of the NUCB1 protein, is a determinant of its export from the ER. In fact, single amino acid substitution (P28A) led to predominant ER distribution and reduced the secretion of NUCB1 without affecting its maturation process in the ER. We also demonstrated that Pro⁺² is required for concentration at the ERES. It is important to note that Pro⁺² was also conserved functionally in other proteins. Our results indicate that Pro⁺² can function as a new ER export signal.     

Vesicular Inhibitory Amino Acid Transporter Is a Cl?/γ-Aminobutyrate Co-transporter

The vesicular inhibitory amino acid transporter (VIAAT) is a synaptic vesicle protein responsible for the vesicular storage of γ-aminobutyrate (GABA) and glycine which plays an essential role in GABAergic and glycinergic neurotransmission. The transport mechanism of VIAAT remains largely unknown. Here, we show that proteoliposomes containing purified VIAAT actively took up GABA upon formation of membrane potential (Δψ) (positive inside) but not ΔpH. VIAAT-mediated GABA uptake had an absolute requirement for Cl⁻ and actually accompanied Cl⁻ movement. Kinetic analysis indicated that one GABA molecule and two Cl⁻ equivalents were transported during one transport cycle. VIAAT in which Glu²¹³ was specifically mutated to alanine completely lost the ability to take up both GABA and Cl⁻. Essentially the same results were obtained with glycine, another substrate of VIAAT. These results demonstrated that VIAAT is a vesicular Cl⁻ transporter that co-transports Cl⁻ with GABA or glycine in a Δψ dependent manner. It is concluded that Cl⁻ plays an essential role in vesicular storage of GABA and glycine.     

Continuous 2-keto-<Emphasis Type="SmallCaps">l</Emphasis>-gulonic acid fermentation from <Emphasis Type="SmallCaps">l</Emphasis>-sorbose by <Emphasis Type="Italic">Ketogulonigenium vulgare</Emphasis> DSM 4025

A single-stage continuous fermentation process for the production of 2-keto-l-gulonic acid (2KGA) from l-sorbose using Ketogulonigenium vulgare DSM 4025 was developed. The chemostat culture with the dilution rate that was calculated based on the relationship between the 2KGA production rate and the 2KGA concentration was feasible for production with high concentration of 2KGA. In this system, 112.2 g/L of 2KGA on the average was continuously produced from 114 g/L of l-sorbose. A steady state of the fermentation was maintained for the duration of more than 110 h. The dilution rate was kept in the range of 0.035 and 0.043 h⁻¹, and the 2KGA productivity was 3.90 to 4.80 g/L/h. The average molar conversion yield of 2KGA from l-sorbose was 91.3%. Under the optimal conditions, l-sorbose concentration was kept at 0 g/L. Meanwhile, the dissolved oxygen level was changing in response to the dilution rate and 2KGA concentration. In the dissolved oxygen (DO) range of 16% to 58%, it was revealed that the relationship between DO and D possessed high degree of positive correlation under the l-sorbose limiting condition (complete consumption of l-sorbose). Increasing D closer to the critical value for washing out point of the continuous fermentation, DO value tended to be gradually increased up to 58%. In conclusion, an efficient and reproducible continuous fermentation process for 2KGA production by K. vulgare DSM 4025 could be developed using a medium containing baker’s yeast without using a second helper microorganism.     

Synthesis and characterization of platinum(II) complexes of 2N1O-donor ligands with a pendent indole ring

The Pt(II) complexes of 2N1O-donor ligands containing a pendent indole, 3-[N-2-pyridylmethyl-N-2-hydroxy-3,5-di(tert-butyl)benzylamino]ethylindole (Htbu-iepp), and 1-methyl-3-[N-2-pyridylmethyl-N-2-hydroxy-3,5-di(tert-butyl)benzylamino]ethylindole (Htbu-miepp) (H denotes an ionizable hydrogen), were synthesized, and the structure of [Pt(tbu-iepp)Cl] (1) was determined by X-ray analysis. Complex 1 prepared in CH₃CN was revealed to have the C2 atom of the indole ring bound to Pt(II) with the Pt(II)-C2 distance of 1.981(3) Å. On the other hand, [Pt(tbu-miepp)Cl] (2) was concluded to have a phenolate coordination instead of the C2 atom of the indole ring by ¹H NMR spectra. Reaction of 1 with 1 equiv. of Ce(IV) in DMF gave the corresponding one-electron oxidized species, which exhibited an ESR signal at g = 2.004 and an absorption peak at 567 nm, indicating the formation of the Pt(II)-indole-π-cation radical species. The half-life, t_1/2, of the radical species at −60 °C was calculated to be 43 s (k_obs = 1.6 × 10⁻² s⁻¹).     

Association of a polymorphism of the apolipoprotein E gene with chronic kidney disease in Japanese individuals with metabolic syndrome

The purpose of the present study was to identify genetic variants that confer susceptibility to chronic kidney disease (CKD) in Japanese individuals with metabolic syndrome. The study population comprised 2150 Japanese individuals with metabolic syndrome, including 411 subjects with CKD [estimated glomerular filtration rate (eGFR) < 50 mL/min/1.73m²] and 1739 controls (eGFR ≥ 60 mL/min/1.73m²). The genotypes for 100 polymorphisms of 80 candidate genes were determined. The chi-square test, multivariable logistic regression analysis with adjustment for covariates, as well as a stepwise forward selection procedure revealed that nine polymorphisms of APOE, ABCA1, PTGS1, TNF, CPB2, AGTR1, OR13G1, and GNB3 were associated (P < 0.05) with the prevalence of CKD. Among these polymorphisms, the ? 219G → T polymorphism of APOE (rs405509) was most significantly associated with CKD in Japanese individuals with metabolic syndrome.