Adult rat hepatocytes cultured on an oxygen-permeable film increases the activity of albumin secretion

Primary culture of rat hepatocyte was performed in an oxygen-permeable film dish (F-dish), which would be expected to give an oxygen-rich culture condition. In the conventional culture dish in which the depth of medium was 2 mm, the oxygen tension (pO₂) in the medium decreased from 19% (144 mmHg) to 0.3% (2.3 mmHg) within 2 hr, while the pO₂ in the F-dish maintained 8.5% (64.6 mmHg) even after 2 hr. The adverse effect of the oxygen-deficiency appeared in the albumin secretion activity of the hepatocytes and it was more remarkable in the early period of culture. The average rate of albumin secretion for the initial 48 hr was 2.0 μg ml^-1 hr^-1 or 96 μg 10⁶ cells^-1 day^-1 in the F-dish. The average rate of albumin secretion for the initial 12 hr was only 0.36 μg ml^-1 hr^-1 in the conventional culture dish. The activity of ammonia elimination in the F-dish was 20–50% higher than the conventional culture dish. Three-dimensional aggregate was formed only in the F-dish. The advantage of three-dimensional aggregate for albumin secretion was not clear compared with two-dimensional monolayer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Heterologous production of a new lasso peptide brevunsin in <Emphasis Type="Italic">Sphingomonas subterranea</Emphasis>

A shuttle vector pHSG396Sp was constructed to perform gene expression using Sphingomonas subterranea as a host. A new lasso peptide biosynthetic gene cluster, derived from Brevundimonas diminuta, was amplified by PCR and integrated to afford a expression vector pHSG396Sp-12697L. The new lasso peptide brevunsin was successfully produced by S. subterranea, harboring the expression vector, with a high production yield (10.2 mg from 1 L culture). The chemical structure of brevunsin was established by NMR and MS/MS experiments. Based on the information obtained from the NOE experiment, the three-dimensional structure of brevunsin was determined, which indicated that brevunsin possessed a typical lasso structure. This expression vector system provides a new heterologous production method for unexplored lasso peptides that are encoded by bacterial genomes.     

Novel glycoproteins inhibiting the binding of colorectal cancer cells to E-selectin

Novel glycoproteins carrying sialyl-LeA (SLeA) antigens (SL-GP) were isolated from ascites fluid from a patient with colorectal cancer by immunoaffinity chromatography. Their characteristics, including binding capacity to E-selectin, were investigated. SL-GP showed a typical mucin type amino acid composition in which Ser, Thr and Pro together accounted for greater than 50% of the total amino acid residues. A large amount of carbohydrate (about 80%) was present in SL-GP. The number of O-glycans carrying SLeA antigens comprised about 9% of the total number of O-glycosidic chains. SL-GP could bind to IL-1 treated HUVEC, and the binding was inhibited by anti-E-selectin and anti-SLeA monoclonal antibodies. The binding of colorectal cancer cells, LS 180, to HUVEC was assayed in the presence of SL-GP, oligosaccharides prepared from SL-GP and human milk SLeA hexasaccharide. SL-GP inhibited the binding most effectively, whereas equivalent amounts of the SL-GP oligosaccharides and milk SLeA hexasaccharide inhibited it only slightly. These results constitute direct evidence that a unique arrangement of SLeA antigens on the polypeptide chain, probably a cluster, is essential for the binding to E-selectin.     

Purification and Characteristics of an Autolytic Chitinase of Piromyces communis OTS1 from Culture Medium

An autolysis chitinase was purified from the cultural medium of the anaerobic fungus Piromyces communis OTS1 by ammonium sulfate precipitation, affinity chromatography with regenerated chitin, chromato-focusing, gel filtration, and chromato-focusing again. The optimal pH and temperature were 6.0 and 50°C, respectively, for a 20-min assay. The chitinase was stable from pH 6.0 to 8.0, but was unstable at 70°C for 20 min. The molecular mass of chitinase was estimated by SDS-PAGE to be 44.9 kDa, and its pI was 4.4. The enzyme activity, which was of the ‘endo’ type, was inhibited by Hg²⁺ and allosamidin. The chitinase hydrolyzes chitin powder and fungal cell walls at a higher rate than an artificial chitin substrate. It can be concluded that extracellular chitinase is similar to cytosolic chitinase, but they are not the same protein. Received: 3 December 1996 / Accepted: 28 January 1997     

Phytochrome Participation in Induction of Haustoria in Cuscuta japonica, a Holoparasitic Flowering Plant

Seedlings of Cuscuta japonica pasted on an acrylic plate developedmany haustoria in response to far-red light after pre-irradiationwith white light. The effect of far-red light was cancelledcompletely by red light irradiated immediately after the far-redlight. In order to elucidate the photoreceptor(s) for photocontrolin the induction of haustoria, action spectra for the photo-inductionand its reversion were determined in the wavelength region from340 nm to 800 nm using the Okazaki Large Spectrograph. The action spectrum for the induction of haustoria had a pronouncedpeak at 740 nm and a small peak at 420 nm, while the actionspectrum for reversal of the induction had a pronounced peakat 660 nm and a small peak at 380 nm. These results indicatethat phytochrome is involved in the photocontrol of inductionof haustoria in Cuscuta japonica. Considering the far-red /redreversibility, it was suggested that phytochrome B is the photorecepter.This is the first reliable evidence of phytochome participationon development of haustoria in parasitic plants. ⁴Present address: Graduate School of Science Division of BiologicalScience, Nagoya University, Nagoya, 464-01 Japan.     

Characteristics of rabbit ClC-2 current expressed in Xenopus oocytes and its contribution to volume regulation

In theXenopus oocyte heterologous expressionsystem, the electrophysiological characteristics of rabbit ClC-2current and its contribution to volume regulation were examined.Expressed currents on oocytes were recorded with a two-electrodevoltage-clamp technique. Oocyte volume was assessed by taking picturesof oocytes with a magnification of ×40. Rabbit ClC-2 currentsexhibited inward rectification and had a halide anion permeabilitysequence of Cl  Br  I  F. ClC-2 currents wereinhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),diphenylamine-2-carboxylic acid (DPC), and anthracene-9-carboxylic acid(9-AC), with a potency order of NPPB > DPC = 9-AC, but were resistant to stilbene disulfonates. These characteristics are similarto those of rat ClC-2, suggesting rabbit ClC-2 as a counterpart of ratClC-2. During a 30-min perfusion with hyposmolar solution, currentamplitude at 160 mV and oocyte diameter were compared amongthree groups: oocytes injected with distilled water, oocytes injectedwith ClC-2 cRNA, and oocytes injected with ClC-2NT cRNA (an openchannel mutant with NH₂-terminaltruncation). Maximum inward current was largest in ClC-2NT-injectedoocytes (5.9 ± 0.4 µA), followed by ClC-2-injected oocytes(4.3 ± 0.6 µA), and smallest in water-injected oocytes(0.2 ± 0.2 µA), whereas the order of increase in oocytediameter was as follows: water-injected oocytes (9.0 ± 0.2%) > ClC-2-injected oocytes (5.3 ± 0.5%) > ClC-2NT-injected oocytes (1.1 ± 0.2%). The findings that oocyte swelling wassmallest in oocytes with the largest expressed currents suggest thatClC-2 currents expressed in Xenopusoocytes appear to act for volume regulation when exposed to ahyposmolar environment.

     

Epidemiological analysis of serum anti‐Pseudomonas aeruginosa PcrV titers in adults

Of the various virulence mechanisms of the opportunistic pathogen Pseudomonas aeruginosa, the type III secretion system (TTSS) has been characterized as a major factor associated with acute lung injury, bacteremia and mortality. In addition, PcrV, a component protein of the TTSS, has been characterized as a protective antigen against infection with P. aeruginosa. This study comprised an epidemiological analysis of serum anti‐PcrV titers in a cohort of Japanese adults. From April 2012 to March 2013, serum anti‐PcrV titers of 198 volunteer participants undergoing anesthesia for scheduled surgeries were measured. The median, minimum and maximum serum anti‐PcrV titers among the 198 participants were 4.09 nM, 1.01 nM and 113.81 nM, respectively. The maximum peaks in the histogram were within the anti‐PcrV 2.00–4.99 nM titer range; values for 115 participants (58.1%) were within this range. Anti‐PcrV titers were more than approximately three‐fold greater (>12 nM) than the median value in 21 participants (10.6%). Ten‐year interval age increases, history of treatment for traffic trauma, and a history of past surgery each showed statistically significant associations with higher anti‐PcrV titers (i.e., >10 nM) than did the other factors assessed by binomial analysis. This study revealed a considerable variation in anti‐PcrV titers in adult subjects without any obvious histories of infection with P. aeruginosa.     

Aurones serve as probes of beta-amyloid plaques in Alzheimer's disease

A novel series of aurone derivatives for in vivo imaging of beta-amyloid plaques in the brain of Alzheimer's disease (AD) were synthesized and characterized. When in vitro binding studies using Abeta(1-42) aggregates were carried out with aurone derivatives, they showed high binding affinities for Abeta(1-42) aggregates at the K(i) values ranging from 1.2 to 6.8 nM. When in vitro plaque labeling was carried out using double transgenic mice brain sections, the aurone derivatives intensely stained beta-amyiloid plaques. Biodistribution studies in normal mice after i.v. injection of the radioiodinated aurones displayed high brain uptake (1.9-4.6% ID/g at 2 min) and rapid clearance from the brain (0.11-0.26% ID/g at 60 min), which is highly desirable for amyloid imaging agents. The results in this study suggest that novel radiolabeled aurones may be useful amyloid imaging agents for detecting beta-amyloid plaques in the brain of AD.