Cytotoxicity of amorphous silica particles against macrophage-like THP-1 cells depends on particle-size and surface properties.

Recent studies have indicated that amorphous silica particles (SPs) show cytotoxicity against various types of cells, including macrophages. However, the mechanism of cell death has not been determined, and systematic investigations of the relationship between particle characteristics and cytotoxicity are still quite limited. Here, we compared the cytotoxicity of SPs of various sizes (30-1000 nm) and surface properties against differentiated THP-1 human macrophage-like cells. We found that 300 and 1000 nm SPs showed cytotoxicity against THP-1 cells, whereas 30, 50, and 70 nm SPs did not induce cell death. We demonstrated that 1000 nm SP showed strong cytotoxicity that depended on reactive oxygen species but was independent of caspases. Furthermore, we showed that surface modification of 1000 nm SPs dramatically suppressed their cytotoxicity. Our results suggest that systematic evaluation of the association between particle characteristics and biological effects is necessary for the creation of safe SPs.

Systemic administration of a PEGylated adenovirus vector with a cancer-specific promoter is effective in a mouse model of metastasis.

Cancer gene therapy by adenovirus vectors (Advs) for metastatic cancer is limited because systemic administration of Adv produces low therapeutic effect and severe side effects. In this study, we generated a dual cancer-specific targeting vector system by using PEGylation and the telomere reverse transcriptase (TERT) promoter and attempted to treat experimental metastases through systemic administration of the vectors. We first optimized the molecular size of PEG and modification ratios used to create PEG-Ads. Systemic administration of PEG-Ad with 20-kDa PEG at a 45% modification ratio (PEG[20K/45%]-Ad) resulted in higher tumor-selective transgene expression than unmodified Adv. Next, we examined the effectiveness against metastases and side effects of a TERT promoter-driven PEG[20K/45%]-Ad containing the herpes simplex virus thymidine kinase (HSVtk) gene (PEG-Ad-TERT/HSVtk). Systemic administration of PEG-Ad-TERT/HSVtk showed superior antitumor effects against metastases with negligible side effects. A cytomegalovirus (CMV) promoter-driven PEG[20K/45%]-Ad also produced antimetastatic effects, but these were accompanied by side effects. Combining PEG-Ad-TERT/HSVtk with etoposide or 5-fluorouracil enhanced the therapeutic effects with negligible side effects. These results suggest that modification with 20-kDa PEG at a 45% modification ratio is the optimal condition for PEGylation of Adv, and PEG-Ad-TERT/HSVtk is a prototype Adv for systemic cancer gene therapy against metastases.

Purification and characterization of coclaurine N-methyltransferase from cultured Coptis japonica cells.

S-Adenosyl-L-methionine (SAM): coclaurine N-methyltransferase (CNMT), which catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the amino group of the tetrahydrobenzylisoquinoline alkaloid coclaurine. was purified 340-fold from Coptis japonica cells in 1% yield to give an almost homogeneous protein. The purified enzyme, which occurred as a homotetramer with a native Mr of 160 kDa (gel-filtration chromatography) and a subunit Mr of 45 kDa (SDS-polyacrylamide gel electrophoresis), had an optimum pH of 7.0 and a pI of 4.2. Whereas (R)-coclaurine was the best substrate for enzyme activity, Coptis CNMT had broad substrate specificity and no stereospecificity CNMT methylated norlaudanosoline, 6,7-dimethoxyl-1,2,3,4-tetrahydroisoquinoline and 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline. The enzyme did not require any metal ion. p-Chloromercuribenzoate and iodoacetamide did not inhibit CNMT activity, but the addition of Co2+, Cu2+ or Mn2+ at 5 mM severely inhibited such activity by 75, 47 and 57%, respectively. The substrate-saturation kinetics of CNMT for norreticuline and SAM were of the typical Michaelis-Menten-type with respective Km values of 0.38 and 0.65 mM.

Bilateral joint replacement of hip and knee joints in patients with rheumatoid arthritis.

Between 1973 and 1981, bilateral total replacement of hip and knee joints was performed in 22 patients with rheumatoid arthritis. During follow-up, 2 patients died of diseases not directly related to the operation. Besides these patients, there were 2 patients who could not be followed up. The remaining 18 patients constituted the subjects for study. We studied the postoperative results, emphasing improvement in the ability to perform everyday activities. Walking ability was improved in 16 patients. There was an increase in the number of patients able to rise from a chair, go up and down stairs, and get in and out of a car, bus or train. Of the 14 married patients (not including 2 patients who were unable to walk and 5 patients over 60 years of age) 7 were able to perform sexual intercourse as well as they had been able to when healthy. The patients who underwent bilateral total replacement of hip and knee joints often presented various problems, for example with regard to indications, the operative technique to use, as well as complications such as heart disease, pulmonary disease, and secondary amyloidosis. We have found, however, that bilateral total replacement of hip and knee joints can accomplish the operative objectives of eliminating pain and improving the quality of daily life in severely handicapped rheumatoid patients.

Molecular characterization of the S-adenosyl-L-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase involved in isoquinoline alkaloid biosynthesis in Coptis japonica.

S-adenosyl-L-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'-OMT) catalyzes the conversion of 3'-hydroxy-N-methylcoclaurine to reticuline, an important intermediate in synthesizing isoquinoline alkaloids. In an earlier step in the biosynthetic pathway to reticuline, another O-methyltransferase, S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransferase (6-OMT), catalyzes methylation of the 6-hydroxyl group of norcoclaurine. We isolated two kinds of cDNA clones that correspond to the internal amino acid sequences of a 6-OMT/4'-OMT preparation from cultured Coptis japonica cells. Heterologously expressed proteins had 6-OMT or 4'-OMT activities, indicative that each cDNA encodes a different enzyme. 4'-OMT was purified using recombinant protein, and its enzymological properties were characterized. It had enzymological characteristics similar to those of 6-OMT; the active enzyme was the dimer of the subunit, no divalent cations were required for activity, and there was inhibition by Fe(2+), Cu(2+), Co(2+), Zn(2+), or Ni(2+), but none by the SH reagent. 4'-OMT clearly had different substrate specificity. It methylated (R,S)-6-O-methylnorlaudanosoline, as well as (R, S)-laudanosoline and (R,S)-norlaudanosoline. Laudanosoline, an N-methylated substrate, was a much better substrate for 4'-OMT than norlaudanosoline. 6-OMT methylated norlaudanosoline and laudanosoline equally. Further characterization of the substrate saturation and product inhibition kinetics indicated that 4'-OMT follows an ordered Bi Bi mechanism, whereas 6-OMT follows a Ping-Pong Bi Bi mechanism. The molecular evolution of these two related O-methyltransferases is discussed.

Metabolic engineering of plant alkaloid biosynthesis.

Plant alkaloids, one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the biosynthetic pathways for scopolamine, nicotine, and berberine have been cloned, making the metabolic engineering of these alkaloids possible. Expression of two branching-point enzymes was engineered: putrescine N-methyltransferase (PMT) in transgenic plants of Atropa belladonna and Nicotiana sylvestris and (S)-scoulerine 9-O-methyltransferase (SMT) in cultured cells of Coptis japonica and Eschscholzia californica. Overexpression of PMT increased the nicotine content in N. sylvestris, whereas suppression of endogenous PMT activity severely decreased the nicotine content and induced abnormal morphologies. Ectopic expression of SMT caused the accumulation of benzylisoquinoline alkaloids in E. californica. The prospects and limitations of engineering plant alkaloid metabolism are discussed.

[99mTc-MAA brain scan and CT in the diagnosis of intracranial diseases (author's transl)].

Brain scan with 99mTc-labelled macroaggregated albumin (MAA brain scan) and computed tomography studies (CT) were performed on 78 patients with intracranial diseases. 99mTc-MAA was injected into right and left carotid artery respectively, at the different time. And then diagnostic evaluation of both techniques were investigated. The MAA brain scan was useful for the detection of ischemic lesion in the early stage after onset of cerebral infarction. It was shown in 29.5% that partial field of anterior cerebral artery was injected through the contralateral artery. In these cases, the MAA brain scan was also useful. In the cases of hemorrhagic lesions, however, the CT was more useful than MAA brain scan. Therefore, the both techniques helped each other in the diagnosis of intracranial diseases.

[Diagnostic evaluation on cerebral hemisphere scintigraphy using 99mTc-MAA (author's transl)].

Using 99mTc-labeled macroaggregated albumin (99mTc-MAA), we made an examination of the cerebral blood flow distribution with various kinds of cerebrovascular diseases in 151 cases. The particle size of MAA used in this study ranged from 10 to 100 micron. Scan was performed after injection of 3 mCi of 99mTc-MAA into common carotid artery. No side effects were observed except for one case with headache. Distribution of 99mTc-MAA was found to be homogeneous in normal cerebral hemisphere but not in the lesion. Most lesion was shown as nonradioactive area. Meningioma with hypervascularity was represented as hot area. By this method cerebral infarct could be easily represented not only in main artery but also in such a very small artery as hardly detected by CAG. This method is therefore useful for detecting cerebral infarct, sequela of cerebral apoplexy, head injury and others. The dose of 99mTc-MAA used in this method is much lower compared with those of common brain scan, its excretion is speedy, and therefore internal radiation exposure is diminished.