A Single Amino Acid Substitution in the Intervening Region of 129K Protein of Cucumber Green Mottle Mosaic Virus Resulted in Attenuated Symptoms.

Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, is a major threat to economically important cucurbit crops worldwide. An attenuated strain (SH33b) derived from a severe strain (SH) of CGMMV caused a reduction in the viral RNA accumulation and the attenuation of symptoms, and it has been successfully used to protect muskmelon plants against severe strains in Japan. In this study, we compared GFP-induced silencing suppression by the 129K protein and the methyltransferase domain plus intervening region (MTIR) of the 129K protein between the SH and SH33b strains, respectively. As a result, silencing suppression activity (SSA) in the GFP-silenced plants was inhibited efficiently by the MTIR and 129K protein of SH strain, and it coincided with drastically reduced accumulation of GFP-specific small interfering RNAs (siRNAs) but not by that of SH33b strain. Furthermore, analyses of siRNA binding capability (SBC) by the MTIR of 129K protein and 129K protein using electrophoretic mobility shift assay revealed that SBC was found with the MTIR and 129K protein of SH but not with that of SH33b, suggesting that a single amino acid mutation (E to G) in the MTIR is responsible for impaired SSA and SBC of SH33b. These data suggest that a single amino acid substitution in the intervening region of 129K protein of CGMMV resulted in attenuated symptoms by affecting RNA silencing suppression.

Lactobacillus gasseri PA-3 directly incorporates purine mononucleotides and utilizes them for growth.

Lactococcus lactis has been reported unable to directly incorporate mononucleotides but instead requires their external dephosphorylation by nucleotidases to the corresponding nucleosides prior to their incorporation. Although Lactobacillus gasseri PA-3 (PA-3), a strain of lactic acid bacteria, has been found to incorporate purine mononucleotides such as adenosine 5'-monophosphate (AMP), it remains unclear whether these bacteria directly incorporate these mononucleotides or incorporate them after dephosphorylation to the corresponding nucleosides. This study evaluated whether PA-3 incorporated radioactively-labeled mononucleotides in the presence or absence of the 5'-nucleotidase inhibitor α,ß-methylene ADP (APCP). PA-3 took up 14C-AMP in the presence of APCP, as well as incorporating 32P-AMP. Furthermore, radioactivity was detected in the RNA/DNA of bacterial cells cultured in the presence of 32P-AMP. Taken together, these findings indicated that PA-3 incorporated purine mononucleotides directly rather than after their dephosphorylation to purine nucleosides and that PA-3 utilizes these purine mononucleotides in the synthesis of RNA and DNA. Although additional studies are required to identify purine mononucleotide transporters in PA-3, this study is the first to show that some lactic acid bacteria directly incorporate purine mononucleotides and use them for growth.

Measurement of the total purine contents and free nucleosides, nucleotides, and purine bases composition in Japanese anchovies (Engraulis japonicus) using high-performance liquid chromatography with UV detection.

Dietary purine restrictions are recommended for patients with hyperuricemia and gout. While measuring the purine contents of various foods in our laboratory using high-performance liquid chromatography (HPLC), we observed and reported changes in purine composition. In this study, we measured the total purine content and free purine of raw anchovies as well as after fermentation, using two methods by HPLC. Method 1 involved acid hydrolysis of all purines, such as nucleic acids and nucleotides, to form four corresponding purine bases. Method 2, which is a non-hydrolysis method, is used to measure the amount of free purines (nucleotide, nucleoside, purine base). As a result of method 1, after fermentation, adenine-related and hypoxanthine-related purines and the total purine levels decreased significantly. Regardless of being raw or fermented, each anchovy contained mainly hypoxanthine- and guanine-related purines. Among the hypoxanthine-related purines, the results of method 2 revealed that the raw anchovies contained a lot of inosine monophosphate (IMP), while after fermentation contained more inosine. In guanine-related and adenine-related purines, those nucleotides decreased by fermentation and nucleosides and bases increased. Measurements of free purines revealed that those reductions after fermentation observed in method 1 were derived from decreased nucleotides. These results indicate that purines are affected by the fermentation bacteria and period.

Species-dependent patterns of incorporation of purine mononucleotides and nucleosides by lactic acid bacteria.

Although most lactic acid bacteria do not directly incorporate purine nucleotides, the strain Lactobacillus gasseri PA-3 was found to incorporate purine mononucleotides. To determine whether the direct uptake of purine mononucleotides is dependent on the species or strain of lactic acid bacteria, incorporation of purine mononucleotides was assessed in L. gasseri, Lactcoccus lactis sbsp. lactis, Streptococcus thermophilus and other species of lactic acid bacteria. Each bacterial strain was incubated with 32P-AMP or 14C-adenosine and the incorporation of each purine was evaluated by measuring their radioactivity. All investigated strains of L. gasseri incorporated 32P-AMP, whereas strains of S. thermophilus and most strains of L. lactis did not. Incorporation of 32P-AMP into strains of Pediococcus was dependent on the strain or species of that genus of bacteria. All investigated strains, except for one strain of L. gasseri, incorporated 14C-adenosine, with S. thermophilus, L. lactis and Pediococcus generally displaying greater incorporation of 14C-adenosine than L. gasseri. Although most lactic acid bacteria such as S. thermophiles and L. lactis do not incorporate purine mononucleotides, some species such as L. gasseri directly incorporate purine mononucleotides. These findings indicate that the preferential incorporation of purine mononucleotides or nucleosides by lactic acid bacteria is dependent on the species or strain.

Simultaneous quantification by HPLC of purines in umami soup stock and evaluation of their effects on extracellular and intracellular purine metabolism.

Ribonucleotide flavor enhancers such as inosine monophosphate (IMP) and guanosine monophosphate (GMP) provide umami taste, similarly to glutamine. Japanese cuisine frequently uses soup stocks containing these nucleotides to enhance umami. We quantified 18 types of purines (nucleotides, nucleosides, and purine bases) in three soup stocks (chicken, consommé, and dried bonito soup). IMP was the most abundant purine in all umami soup stocks, followed by hypoxanthine, inosine, and GMP. The IMP content of dried bonito soup was the highest of the three soup stocks. We also evaluated the effects of these purines on extracellular and intracellular purine metabolism in HepG2 cells after adding each umami soup stock to the cells. An increase in inosine and hypoxanthine was evident 1 h and 4 h after soup stock addition, and a low amount of xanthine and guanosine was observed in the extracellular medium. The addition of chicken soup stock resulted in increased intracellular and extracellular levels of uric acid and guanosine. Purine metabolism may be affected by ingredients present in soups.

The observed variation in the purine composition of food after soaking in sake lees.

In this study, we investigated the alterations in the purine composition of swordfish prepared using a traditional Japanese processing method of soaking in sake lees. These alterations are the byproducts of the yeast fermentation of rice-koji and are renowned for enhancing the umami nature of food. Using a conventional assay method for hydrolyzing all of the purines into four bases and our developed method for simultaneously analyzing purines, we observed the alterations in four purine bases in the soaked sake lees and swordfish. The findings showed that the total purine content, and hypoxanthine-related and guanine-related purines in swordfish decreased after soaking in sake lees. We also analyzed the free purine composition and showed that the ratio of IMP in swordfish was decreased by soaking, while that of inosine in sake lees was increased by soaking swordfish in it.

Specific Binding of the Syringolide Elicitors to a Soluble Protein Fraction from Soybean Leaves.

Syringolides are glycolipid elicitors produced by Gram-negative bacteria expressing Pseudomonas syringae avirulence gene D. The syringolides mediate gene-for-gene complementarity, inducing the hypersensitive response only in soybean plants carrying the Rpg4 disease resistance gene. A site(s) for 125I-syringolide 1 was detected in the soluble protein fraction from soybean leaves, but no evidence for ligand-specific binding to the microsomal fraction was obtained. The Kd value for syringolide 1 binding with the soluble fraction was 8.7 nM, and binding was greatly reduced by prior protease treatment or heating. A native gel assay was also used to demonstrate ligand-specific binding of labeled syringolide 1 with a soluble protein(s). Competition studies with 125I-syringolide 1 and several structural derivatives demonstrated a direct correlation between binding affinity to the soluble fraction and elicitor activity. However, differential competition binding studies disclosed no differences in syringolide binding to soluble fractions from Rpg4/Rpg4 or rpg4/rpg4 soybean leaves. Thus, the observed binding site fulfills several criteria expected of an intracellular receptor for the syringolides, but it is most likely not encoded by the Rpg4 gene. Instead, the Rpg4 gene product may function subsequent to elicitor binding, possibly in intracellular signal transduction.

[The efficacy of perioperative administration of steroid and erythromycin in the surgery for lung cancer complicated with interstitial pneumonia].

OBJECTIVES: We evaluated the efficacy of perioperative administration of steroid and erythromycin in surgery for lung cancer complicated with interstitial pneumonia (IP) to prevent postoperative acute exacerbation. PATIENTS AND METHODS: We operated on 21 lung cancer patients with IP for 10 years. The patients were given 400 mg of erythromycin over 1 week before surgery and re-administered on the 1st operative day. The patients were also given 125 mg of methylprednisolone intravenously just before operation and continued until the 2nd operative day. RESULTS: Lobectomy was performed in 16, segmentectomy or partial resection in 2 each, and completion pneumonectomy in 1. Three patients developed acute exacerbation of IP, but it occurred after the re-operation due to postoperative complications in 2. We experienced no operative death within 30 days, however, 2 died during the hospital stay due to multiple organ failure and sepsis. Seven of 21 patients had postoperative complications; air leakage over 1 week in 4, arrhythmia in 3, and atelectasis, postoperative bleeding, and pneumonia in 1 each, the morbidity rate was 33%. CONCLUSIONS: We conclude that the administration of steroid and erythromycin in surgery for lung cancer with IP was suspected the usefulness to prevent a postoperative acute exacerbation of IP.

Local administration of monoclonal antibody-drug conjugate: a new strategy to reduce the local recurrence of colorectal cancer.

This report investigates the application of monoclonal antibody A7 and its drug conjugate in locally controlling colorectal cancer. The experimental protocol consisted of local retention, lymphatic delivery, normal organ distribution, systemic toxicity, and tumoricidal effects. When 125I-labeled monoclonal antibody (Mab) A7 was injected into the pelvis and the thigh of Balb/c mice, a high local retention unrelated to antigen-antibody interaction was observed at the injected site for 24 h after injection. An analysis of local retension properties related to antigen-antibody interaction, conducted by intratumorally or peritumorally injecting 125I-Mab A7 into the tumor-bearing athymic nude mice, revealed a significantly higher tumor localization of Mab A7 in comparison to i.v. injection. 125I-Mab A7 accumulated to a great extent in the ipsilateral regional lymph node but not in the contralateral regional lymph node. Normal organ accumulation of Mab A7 was lower in the locally injected group than in the i.v. injected group. Intratumoral injection of Mab A7-neocarzinostatin (A7-NCS) led to the complete remission of established tumor in 5 of 6 antigen-positive xenograft-bearing mice but exhibited a complete remission in only 1 of 6 antigen-negative xenograft-bearing mice. A single local injection of A7-NCS inhibited tumor development in 12 of 16 and 5 of 15 antigen-positive tumor-bearing mice and antigen-negative tumor-bearing mice, respectively, whereas neither a systemic injection of A7-NCS and NCS nor a local injection of NCS and saline had a notable inhibitory effect on tumor development. Systemic toxicity of NCS was markedly reduced when it was locally administered in the antibody-conjugated form. These findings indicate that local injection of immunoconjugate is a promising new field for controlling the local recurrence of colorectal cancer.

Evaluation of cellular purine transport and metabolism in the Caco-2 cell using comprehensive high-performance liquid chromatography method for analysis of purines.

Using Caco-2 cells and our previously developed high-performance liquid chromatography method for quantification of purine bases, nucleosides, and nucleotides, we evaluated cellular purine transport and uptake. The analytes were separated using YMC-Triart C18 column with gradient elution. We used Caco-2 cells as intestinal model cells and monitored purine transport across a monolayer for 2 h. The degree of change of purine concentrations in the permeate was very slight; however, it was possible to simultaneously determine these parameters for all purines because of our method's high sensitivity. In the present study, the purine bases (adenine, guanine, hypoxanthine, and xanthine) showed a relatively high permeability as compared with the nucleosides (adenosine, guanosine, inosine, and xanthosine). Increased concentration of metabolites in the permeate was also observed following the addition of purines. In a cell uptake assay, both the cell culture medium (extracellular) and the cells extracted from Caco-2 with acetonitrile:water (7:3) (intracellular) were measured. The additional nucleoside did not increase significantly within the cells. On the other hand, we observed that nucleotide, such as ATP, increased in the cell in a time-dependent manner following the addition of nucleoside. The additional nucleosides were considered to be rather recycled via the salvage pathway than metabolized to purine bases and/or uric acid in the cell. Such differences might have affected the increase in the serum uric acid levels depending on purine form.

Urinary excretion of uric acid, allantoin, and 8-OH-Deoxyguanosine in uricase-knockout mice.

Although uricase-knockout (Uox KO) mice are reported to develop uric acid (UA) nephropathy, those that mature without severe nephropathy could be useful for research into purine metabolism in humans. In this study, we measured the urinary excretion of creatinine, UA, allantoin, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) collected from Uox KO mice housed in metabolic cages. UA and allantoin were determined using liquid chromatography-mass spectrometry and creatinine and 8-OHdG were measured with a commercial kit. Uox KO mice excreted significantly higher levels of UA than wild-type mice (C57BL/6), while the excretion of allantoin was significantly lower. Urinary allantoin was detected in Uox KO mice despite a lack of uricase, which is the same as in humans. In contrast to the elevated levels of UA, the daily excretion of 8-OHdG, an oxidative stress marker, was lower in Uox KO mice. UA is thought to act as an anti-oxidizing agent in humans; thus, these results show that Uox KO mice are potential animal models for research into human purine metabolism.

Evaluation of purine utilization by Lactobacillus gasseri strains with potential to decrease the absorption of food-derived purines in the human intestine.

It is well accepted that frequent and heavy intake of purine-rich foods causes elevation of serum uric acid levels, which is a risk factor of hyperuricemia. Reducing intestinal absorption of dietary purines may attenuate the elevation of serum uric acid levels and exacerbation of hyperuricemia. This reduction may be achieved by the ingestion of lactic acid bacteria that take up purines in the intestine. In this study, we investigated the degree of uptake and utilization of purines of three lactobacilli strains. Among them, Lactobacillus gasseri PA-3 (PA-3) showed the greatest incorporation of 14C-adenine. PA-3 also incorporated 14C-adenosine and 14C-AMP. Additionally, using defined growth medium, PA-3 demonstrated greater proliferation in the presence of these purines than in their absence. Although further investigation is required, ingestion of PA-3 may lower serum uric acid levels by reducing intestinal absorption of purines in humans.

CT simulator: a new 3-D planning and simulating system for radiotherapy: Part 1. Description of system.

A real time CT-linked 3-D treatment planning system, called a CT simulator, has been developed. The basic system consists of a CT scanner, a multi-image display component, a treatment planning device with real time visual optimization, and a laser beam projecting component. All the components are connected on line. The system can be conveniently used for 3-D planning and simulation for radiation therapy within a reasonably short period of time.

CT simulator: a new 3-D planning and simulating system for radiotherapy: Part 2. Clinical application.

We have performed radiotherapy treatment planning (RTP) with a new system called CT simulator in 72 patients. With the system, RTP is performed with the patient lying on the CT couch within a short period of time. All the CT images scanned were immediately transported to the multi-image monitors and to the treatment planning device. Radiotherapy treatment planning could be performed not only at the beam center but also at any CT slice. Using a laser-beam field projector, field outlines were drawn over the patient's skin. In clinical use, the system was useful for cases in which a target lies adjacent to dose limiting organs, cases with a complicated target shape, cases with complicated dose distribution curves, and cases treated with tangential fields. This system enables us to make optimum use of CT information and to make accurate 3-dimensional treatment planning programs.

Targeted chemotherapy in mice with peritoneally disseminated gastric cancer using monoclonal antibody-drug conjugate.

The murine monoclonal antibody A7 (MAb A7) is reactive against most human gastric cancer cell lines. Using a nude mouse peritoneal dissemination model of human gastric cancer, we investigated targeted chemotherapy using a conjugate of neocarzinostatin (NCS) with MAb A7 (A7-NCS). After demonstrating cytotoxicity of the complex against the human gastric cancer cell line MKN45 in vitro, we intraperitoneally injected A7-NCS, NCS or saline into nude mice bearing peritoneally disseminated human gastric cancer. A7-NCS inhibited peritoneal dissemination significantly more effectively than NCS. MAb A7 may prove to be an effective carrier for antineoplastic drugs in patients with peritoneal dissemination of gastric cancer.

Changes in expression of the antigen recognized by monoclonal antibody A7 in human pancreatic carcinoma cells following exposure to anticancer agents.

Techniques which can increase the expression level of tumor-associated antigens may improve immunotargeting therapy. We studied the reactivity of MAb A7 toward an antigen expressed on the surface of the human pancreatic cancer cell line HPC-YS after treatment with various antitumoral agents. When we applied 1 microg/ml mitomycin C (MMC) or 0.1 microg/ml neocarzinostatin (NCS) for 1 h, A7 recognizing antigen expression was enhanced until 24 h after the treatments. At a dose that completely suppressed cell growth, increased antigen expression was maintained for 96 h. Therefore, this study suggests that the combined application of an anticancer drug and MAb A7 may be useful for immunotargeting chemotherapy.

Bioavailability of 50- and 75-mg oral etoposide in lung cancer patients.

This study was designed to determine the bioavailability of etoposide capsules administered orally at doses of 50 and 75 mg. Patients with inoperable or relapsed lung cancer, who had an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2 and adequate organ function, were eligible. A group of 17 patients were evaluable, all of whom were 75 years old or less, with an ECOG performance status of 0 or 1. The bioavailability of oral etoposide was determined by measuring the area under the etoposide plasma concentration versus time curve (AUC) on days 1, 10 and 21 during a once-daily regimen of oral administration for 21 consecutive days and comparing the value with the AUC achieved following intravenous administration 1 or 2 weeks after the last oral dose. The bioavailability of 50, 75 and 100 mg oral etoposide was determined in six, nine and two patients, respectively. The mean etoposide bioavailabilities (+/- SD) of the 50-mg and 75-mg doses were 47 +/- 11% and 59 +/- 18%, respectively, and of the 100-mg dose in two patients were 51% and 33%, respectively. There was no statistically significant difference in bioavailability between the 50-mg and 75-mg doses. The bioavailability of low-dose oral etoposide was the same as that reported in previous higher dose oral etoposide bioavailability studies and that shown on the package insert supplied by the manufacturer. Improved bioavailability of low-dose oral etoposide was therefore not observed in a population of Japanese patients.

Salivary drug monitoring of irinotecan and its active metabolite in cancer patients.

To assess the clinical usefulness of salivary monitoring of irinotecan (CPT-11) and its active metabolite (SN-38), we examined the clinical pharmacological profile of both drugs in 9 patients with thoracic malignancies who received 60 mg/m2 CPT-11 (21 courses). Plasma and unstimulated whole saliva were collected over a 24-h period, and concentrations of CPT-11 and SN-38 were measured by high-performance liquid chromatography. Both CPT-11 and SN-38 were detectable in saliva, and the concentration-time curves in plasma and saliva showed a very similar pattern. A good correlation was observed between the saliva concentration (C3) and the plasma concentration (Cp) for both CPT-11 and SN-38 (r = 0.732, P < 0.0001 and r = 0.611, P < 0.0001, respectively). The area under the concentration-time curve calculated for saliva (AUCs) correlated with that generated for plasma (AUCp) for both CPT-11 and SN-38 (r = 0.531, P = 0.012 and r = 0.611, P = 0.0025, respectively). These results suggest that it may be feasible to use saliva instead of plasma for pharmacokinetics/pharmacodynamics studies of CPT-11.

Biodistribution of murine and chimeric Fab fragments of the monoclonal antibody A7 in human pancreatic cancer.

Much recent research has focused on the use of monoclonal antibodies (MAbs) in the immunodetection of solid tumors. Fab fragments of MAbs are more suitable for immunoscintigraphy than intact MAbs. Recently, human-mouse chimeric antibodies have been developed in an effort to reduce human antimouse antibody (HAMA) production by murine MAbs in humans. In this study, 125I-labeled murine and chimeric Fab fragments of the MAb A7 were injected i.v. into nude mice bearing a human pancreatic cancer (HPC-YS) xenograft. The radioactivity in tumors and in normal tissues was subsequently measured. The tumor tissue/blood ratio (T/B) of 125I-labeled murine and chimeric Fab fragments of MAb A7 increased with time in a similar manner and reached 9.68 +/- 2.54 and 10.49 +/- 1.50, respectively, 24 h after injection. Moreover, the T/Bs of 125I-labeled murine and chimeric Fab fragments of MAb A7 were greater than the T/B of intact MAb A7. When mice bearing tumors that did not react with MAb A7 were studied, 125I-labeled murine and chimeric Fab fragments did not localize specifically to the tumors. These results suggests that chimeric Fab fragments of MAb A7 are useful carriers of radionuclides for the immunodetection of human pancreatic cancer, with equivalent activity to murine Fab fragments and less theoretical potential to induce a HAMA response.

Partial deficiency of hypoxanthine-guanine phosphoribosyltransferase manifesting as acute renal damage.

A 32-year-old man who had had frequent gouty arthritis over the past 17 years, was admitted for acute renal failure. Acute renal failure was improved rapidly after medication was resumed and the patient was sufficiently hydrated. The hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity in the patient had been reduced to about 30% of the normal control. Therefore we considered that this patient suffered from a partial deficiency of HPRT. A point mutation of HPRT gene 68G (guanine) to T (thymine) was detected. This is a mutation that has not been previously reported. Familial analysis indicated that his mother and sister were heterozygotes.