Adoptive immunotherapy using immobilized anti-CD3 mAb-activated autologous lymphocytes: the strong cytotoxicity was supported by CD16+ cells which proliferated in prolonged cultures]

Inflammatory cytokines are able to facilitate the homing of transferred lymphocytes, tumor cell lysis through induction of adhesion molecules, also able to reduce tumor cell susceptibility to LAK cells by increasing tumor cell class I antigen. Investigation with 12 cell lines suggested that promotion of lysis by ICAM-1 was more responsible than protection by (allogeneic) class I Ags (Fig. 1). PBMC were cultured in anti-CD3 coated flasks with rIL-2. CD3+ cells dominated until day 7, decreased thereafter with CD4+. CD8+ and CD16+ increased (Fig. 2). Strong cytotoxicity obtained in some cultures correlated well with CD16+, contributing exclusively among several variables to the activity estimation in multiple regression analysis (Fig. 4). Among 6 cases, in which 2 or more cycles of transfer was done, 1 was prophylaxis of recurrence, in 2 of 3 advanced metastasis cases in which cells were transferred as BRM in the course of chemotherapy, survival of half a year was obtained in good QOL with suppressed disease and adequate level of PBL number. In 2 other cases, inflammation eliciting local treatments were combined. In the case 4, three large liver metastasis from colon cancer which resisted topical ethanol injection and chemotherapy, responded to the transfer with reduced lesions to 1/8 (Fig. 8). In the case 5, abdominal metastasis from colon cancer were removed, liver metastasis were injected of ethanol, and cells were transferred. Responses were obtained to immunotherapy in a certain degree, while never to any chemotherapy.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

A chromosomal method to distinguish between X- and Y-bearing spermatozoa of the bull in zona-free hamster ova

The interspecific in-vitro fertilization system using zona-free hamster ova was adopted to distinguish chromosomally between X- and Y-bearing bull spermatozoa. Frozen semen samples were thawed and washed with modified BWW medium including 0.3% BSA (pH 7.2) to remove cryoprotective medium. Motile spermatozoa were recovered from the semen by the 'swim-up' method. These spermatozoa were treated with ionophore A23187 to facilitate capacitation. Adequate capacitation of spermatozoa was found by their movement patterns and the insemination was performed at the optimum time. The fertilized ova were cultured in Medium TC199 with 10% FBS including podophyllotoxin and vinblastine until they reached first cleavage metaphase. Chromosome slides were prepared by our gradual fixation-air drying method. The success rate of the method was 56% of the number corresponding to the zona-free ova used. Altogether 1116 spermatozoa from 4 different bulls were successfully analysed and the ratio of X- and Y-bearing spermatozoa was 542:574 (P greater than 0.3).     

Complete nucleotide sequence of the mitochondrial DNA from a liverwort,Marchantia polymorpha

Libraries of cosmid and plasmid clones covering the entire region of mtDNA from the liverwortMarchantia polymorpha were constructed. These clones were used for the determination of the complete nucleotide sequence of the liverwort mtDNA totally 186,608 bp (GenBank no. M68929) and including genes for 3 species of ribosomal RNAs, 29 genes for 27 species of transfer RNAs, and 30 genes for functionally known proteins (16 ribosomal proteins, 3 subunits of cytochromec oxidase, apocytochromeb protein, 3 subunits of H⁺-ATPase, and 7 subunits of NADH ubiquinone oxidoreductase). The genome also contains 32 unidentified open reading frames. Thus the complete nucleotide sequences from both chloroplast and mitochondrial genomes have been determined in the same organism. Plasmid clones are available upon the request. Gene names are represented according to Lonsdale and Leaver (1988) with modifications recommended by Lonsdale (personal communication).     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.     

The induction of recessive mutations in mouse primordial germ cells with N-ethyl-N-nitrosourea

A specific-locus test was carried out to examine the mutagenic activity of N-ethyl-N-nitrosourea (ENU) on mouse primordial germ cells (PGC). Embryos of C3H/He mice were treated transplacentally with 30 or 50 mg ENU per kg of maternal body weight on day 8.5, 10.5, or 13.5 of gestation (G8.5 day, G10.5 day, or G13.5 day). Male and female mice that had been treated with ENU in embryonic stages were mated with female or male tester PW mice to detect recessive mutations induced in PGC.

ENU induced recessive mutations at a relatively high rate in PGC at these developmental stages. The most sensitive stage was G10.5 day. On G8.5 day, the induced mutation rate in males and females was not significantly different. Cluster mutations, which originate from the limited number of PGC and cell killing, were more frequently induced at an earlier developmental stage. The induced mutation rate per unit dose of ENU (1 mg/kg) was higher in G8.5 and G10.5 day PGC than in stem-cell spermatogonia. It can be concluded that mouse PGC are more sensitive than stem-cell spermatogonia to the induction of recessive mutations by ENU.     

Effects of hydrostatic pressure on the ultrastructure and leakage of internal substances in the yeast Saccharomyces cerevisiae

The structural damage to and leakage of internal substances from Saccharomyces cerevisiae 0–39 cells induced by hydrostatic pressure were investigated. By scanning electron microscopy, yeast cells treated at room temperature with pressuresbellw 400 MPa for 10 min showed a slight alteration in outer shape. Transmission electron microscopy, however, showed that the inner structure of the cell began to be affected, especially the nuclear membrane, when treated with hydrostatic pressure around 100 MPa at room temperature for 10 min; at more than 400–600 MPa, further alterations appeared in the mitochondria and cytoplasm. Furthermore, when high pressure treatment was carried out at — 20° C, the inner structure of the cells was severely damaged even at 200 MPa, and almost all of the nuclear membrane disappeared, although the fluorescent nucleus in the cytoplasm was visible by 4,6-diamidino-2-phenylindole (DAPI) staining. The structural damage of pressure-treated cells was accompanied by the leakage of internal substances. The efflux of UV-absorbing substances including amino acid pools, peptides, and metal ions increased with increase in pressure up to 600 MPa. In particular, amounts of individual metal ion release varied with the magnitude of hydrostatic pressures over 300 MPa, which suggests that the ions can be removed from the yeast cells separately by hydrostatic pressure treatment. Correspondence to: S. Shimada     

Hamster pulmonary endocrine cells with positive immunostaining for calbindin-D28K

 We have examined the distribution of calcium-binding proteins (CaBPs) in adult and fetal lungs of Syrian golden hamsters (Mesocricetus auratus) using immunostaining with confocal laser microscopy and electron microscopy. Single and grouped (neuroepithelial body; NEB) endocrine cells were distributed from bronchi to alveolar ducts in the adult lung. Serial frozen sections immunostained for CaBPs in combination with immunostaining for endocrine markers such as calcitonin gene-related peptide, serotonin, PGP9.5, and synaptophysin revealed that positive immunostaining for calbindin-D_28K (CB-D28K) was seen in single endocrine cells and NEBs. However, other so-called EF-hand family CaBPs, parvalbumin and calretinin, were not detected. Electron microscopically, positive immunoreaction for CB-D28K was mainly in the organelle-free cytoplasmic matrix of endocrine cells, and partly in nuclei and associated with secretory granules and endoplasmic reticulum. In fetal developing lungs, endocrine cells appeared first on gestational day 13, and they were positive for all the endocrine markers used. However, pulmonary endocrine cells were positively immunostained for CB-D28K from gestational days 15 and 16 onward. In summary, our observations suggest that CB-D28K is a useful marker for endocrine cells of the lung, and CB-D28K could function as a mediator of endocrine stimulation or calcium homeostasis in pulmonary endocrine cells. Accepted: 17 June 1997     

Purification and properties of a lytic enzyme from the cell wall of Chlorella ellipsoidea C-87

Cell wall lytic activity was detected in the culture medium and cell wall of 1AM Chlorella ellipsoidea C-87. The enzymes of both fractions had their highest activity at pH 5. The lytic activity bound to the cell wall consisted of a polysaccharide releasing enzyme, an exo-type enzyme releasing disaccharide, and glucosidase; but only the polysaccharide releasing enzyme was solubilized by lithium chloride. A polysaccharide releasing enzyme with a molecular weight around 40 kDa was isolated from the culture medium. Hemicellulose is degraded by the polysaccharide releasing enzyme, and the rigid wall by the exo-type enzyme.