Phosphate Induces Rapid H2O2 Generation in Soybean Suspension Cells

Abstract: Involvement of reactive oxygen species has been implicated in plant defence against pathogens. We report here a novel pathway of H₂O₂ generation induced by the addition of phosphate in soybean ( Glycine max L.) cell suspension cultures. This H₂O₂ generation was initiated shortly after the addition of phosphate, and lasted only approximately one hour, as opposed to several hours observed during an attack by an avirulent strain of the bacterial pathogen Pseudomonas syringae pv. glycinea (Psg). In addition, when cell cultures were treated with both phosphate and the avirulent pathogen, two distinct oxidative burst events were observed. In contrast to DPI-sensitive Psg -induced H₂O₂ generation, phosphate-induced H₂O₂ generation was insensitive to this NADPH oxidase inhibitor. This suggests that an NADPH oxidase-independent pathway may be involved in the phosphate-induced H₂O₂ accumulation, which could be involved in sensing of phosphate availability in the environment.     

Novel and recurrent COMP (cartilage oligomeric matrix protein) mutations in pseudoachondroplasia and multiple epiphyseal dysplasia

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are common skeletal dysplasias with impaired enchondral ossification and premature degenerative joint disease. The two disorders were in the past considered to be distinct clinical entities; however, recent studies have proven that both diseases can result from mutations of the gene encoding cartilage oligomeric matrix protein (COMP). To characterize further COMP mutations and investigate phenotype-genotype relationships, we screened this gene in 15 patients with PSACH or MED by directly sequencing polymerase chain reaction products from genomic DNA. We identified ten mutations involving conserved residues among the eight calmodulin-like repeats of the gene product: seven were novel missense mutations in exons 9, 10, 11, 13 or 14, and the other three resulted from deletion of one of the five GAC repeats in exon 13. We have found that the GAC repeats in the 7th calmodulin-like repeat in exon 13 represent a hot-spot for mutation, and that mutations in the 7th calmodulin-like repeat produce severe PSACH phenotypes while mutations elsewhere in the gene exhibit mild PSACH or MED phenotypes. These genotype-phenotype correlations may facilitate molecular diagnosis and classification of PSACH and MED, and provide insight into the relationship between structure and function of the COMP gene product. Received: 1 June 1998 / Accepted: 22 October 1998     

A new human hepatocellular carcinoma cell line (KYN-1) with a transformation to adenocarcinoma

Summary A human hepatocellular carcinoma (HCC) cell line (KYN-1) has been established from a resected HCC of a 58-yr-old Japanese, male patient with HCC. Original resected HCC was moderately differentiated and proliferated in a solid pattern with vague trabecular structure in part. This cell line has been maintained for 10 mo. through 50 passages. Morphological features of KYN-1 cells demonstrated one or more large, round-to-oval nuceli with prominent nucleoli and eosinophilic polygonal-to-spindle abundant cytoplasm. In addition, some of these cells contained mucicarmin-positive materials in the cytoplasm. The cells exhibited a typical epithelial feature with pavementlike cell arrangement, and lacked contact inhibition. The doubling times of the cells grown in a serum-containing and a serum-fre, medium were about 31 h and 10 to 11 d, respectively. Functonally, KYN-1 cells produced albumin, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), ferritin, β₂-microglobulin (BMG), and α₁-anti-trypsin (AAT). Positive reactions for albumin, AFP, CEA, and ferritin were identified in the cells by immunohistochemical techniques. Chromosome study revealed the chromosome number in a range from 61 to 74 without mode. The tumorigenicity of KYN-1 cells was identified by the tumor formation after subcutaneous inoculation of the cells into nude mice. The developed tumor showed compact growth of the tumor cells with gland formations containing mucicarmin-positive materials. Features of adenocarcinoma were identified by electron microscopy. The tumor cells were also identified to contain albumin, AFP, CEA, ferritin, and AAT by immunohistochemical techniques. AFP, CEA, and BMG were detected in the sera of nude mice. Thus, KYN-1 cells represented the morphologic features of adenocarcinoma, retaining some characteristics of original HCC. These findings suggest that KYN-1 is a new human HCC cell line with transformation to adenocarcinoma, which will provide useful information to clarify the histogenesis of combined hepatocellular and cholangiocellular carcinoma. This study was supported in part by the Sarah Cousins Fund.     

The cloned β-mannanase gene from alkalophilic Bacillus sp. AM-001 produces two β-mannanases in Escherichia coli

The gene encoding -mannanase was cloned from alkalophilic Bacillus sp. AM-001 into Escherichia coli JM 101 by inserting HindIII-generated DNA fragments into the HindIII site of pUC19. A 2.0 kb XbaI-PstI fragment of the donor strain DNA was sufficient for -mannanase synthesis. The amount of -mannanase expressed in E. coli JM101 harboring pMAH3 (containing a 2.4 kb XbaI-HindIII fragment) was about 24% of the activity produced by the donor strain. E. coli JM101 harboring pMAH3 was found to produce two enzymatically active -mannanases (A and B). These two -mannanases were purified to electrophoretically homogenous states. The -mannanase A had enzymatic properties similar to those of the -mannanases M-I and M-II produced by alkalophilic Bacillus sp. AM-001, and the -mannanase B resembled its -mannanase M-III. In contrast to -mannanase production in the donor strain, that in E. coli was not inducible. The NH₂-terminal amino acid sequences from amino acid 1 (Asn) to 9 (Gln) of the three -mannanases purified from alkalophilic Bacillus sp. AM-001 coincide with those from amino acid 4 (Asn) to 12 (Gln) of the two -mannanases purified from E. coli transformant.     

Analysis of Plasma Protein Concentrations and Enzyme Activities in Cattle within the Ex-Evacuation Zone of the Fukushima Daiichi Nuclear Plant Accident

The effect of the Fukushima Daiichi Nuclear Power Plant (FNPP) accident on humans and the environment is a global concern. We performed biochemical analyses of plasma from 49 Japanese Black cattle that were euthanized in the ex-evacuation zone set within a 20-km radius of FNPP. Among radionuclides attributable to the FNPP accident, germanium gamma-ray spectrometry detected photopeaks only from ¹³⁴Cs and ¹³⁷Cs (radiocesium) commonly in the organs and in soil examined. Radioactivity concentration of radiocesium was the highest in skeletal muscles. Assuming that the animal body was composed of only skeletal muscles, the median of internal dose rate from radiocesium was 12.5 μGy/day (ranging from 1.6 to 33.9 μGy/day). The median of external dose rate calculating from the place the cattle were caught was 18.8 μGy/day (6.0–133.4 μGy/day). The median of internal and external (total) dose rate of the individual cattle was 26.9 μGy/day (9.1–155.1 μGy/day). Plasma levels of malondialdehyde and superoxide dismutase activity were positively and glutathione peroxidase activity was negatively correlated with internal dose rate. Plasma alanine transaminase activity and percent activity of lactate dehydrogenase (LDH)-2, LDH-3 and LDH-4 were positively and LDH-1 was negatively correlated with both internal and total dose rate. These suggest that chronic exposure to low-dose rate of ionizing radiation induces slight stress resulting in modified plasma protein and enzyme levels.     

Sialyltransferase ST3Gal IV deletion protects against temporal lobe epilepsy

Temporal lobe epilepsy (TLE) often becomes refractory, and patients with TLE show a high incidence of psychiatric symptoms, including anxiety and depression. Therefore, it is necessary to identify molecules that were previously unknown to contribute to epilepsy and its associated disorders. We previously found that the sialyltransferase ST3Gal IV is up‐regulated within the neural circuits through which amygdala‐kindling stimulation propagates epileptic seizures. In contrast, this study demonstrated that kindling stimulation failed to evoke epileptic seizures in ST3Gal IV‐deficient mice. Furthermore, approximately 80% of these mice failed to show tonic–clonic seizures with stimulation, whereas all littermate wild‐type mice showed tonic–clonic seizures. This indicates that the loss of ST3Gal IV does not cause TLE in mice. Meanwhile, ST3Gal IV‐deficient mice exhibited decreased acclimation in the open field test, increased immobility in the forced swim test, enhanced freezing during delay auditory fear conditioning, and sleep disturbances. Thus, the loss of ST3Gal IV modulates anxiety‐related behaviors. These findings indicate that ST3Gal IV is a key molecule in the mechanisms underlying anxiety – a side effect of TLE – and may therefore also be an effective target for treating epilepsy, acting through the same circuits.

     

A historical view of schistosomiasis japonica in the Chikugo river basin. What can we learn from autopsy?

Three hundred and forty-nine autopsy cases of schistosomiasis japonica were divided into two groups, based on the pathomorphology. Frequent regressive hepatic lesions such as active schistosomal lesion and destruction of limiting plates characterized the first group. The second group showed reparative hepatic lesions such as regeneration of the collapsed parenchyma, newly formed limiting plates and subsequent narrowing and disappearance of fibrous septa. Complications of liver cirrhosis and hepatocellular carcinoma related to viral hepatitis B and/or C also increased. Clonorchiasis was consistently found in both groups.