Cytotoxic cell function and phenotypic analysis of peripheral blood mononuclear cells in cancer patients treated with low-dose interleukin-2 and mitomycin C

We previously found that the ability of peripheral blood mononuclear cells (PBM) of cancer patients to generate lymphokine-activated killer (LAK) cells became remarkably augmented after mitomycin C administration. On the basis of the clinical finding, we designed a treatment regimen comprised of 12 mg/m² mitomycin C i. v. on day 1 and 700 U/m² recombinant interleukin-2 (IL-2) i.v. every 12 h from day 4 through day 8. Of 25 patients with advanced carcinoma, 9 had a partial response and 3 had a minor response. Cytotoxic cell function, including natural killer activity, lymphokine-activated killer (LAK) activity, and the ability to generate LAK cells, and lymphocyte subsets in PBM was measured 1 day before and after either the first or second course of this therapy. The relationship between these parameters and the clinical antitumor response to this treatment was examined. Although the cytotoxic activities were significantly augmented after either the first or second treatment course, no positive correlation was observed between the changes in these cytotoxic activities and the clinical response to this therapy, when patients who either showed a partial response or whose disease remission was partial or minor were defined as responders. Further, phenotypic analysis showed a significant increase in CD2⁺, CD3⁺ CD4⁺ and CD4⁺Leu8^– cells after the firs course, and CD25⁺ cells after either the first or second course of this treatment. The precentages of CD2⁺ and CD25⁺ cells were significantly elevated only in responders but not in nonresponders, suggesting the increase in these subsets was related to clinical response.     

The localized melting of mini-F origin by the combined action of the mini-F initiator protein (RepE) and HU and DnaA of Escherichia coli

Replication of mini-F plasmids requires the initiator protein RepE, which binds specifically to four iterons within the origin (ori2), as well as some host factors that are involved in chromosomal DNA replication. To understand the role of host factors and RepE in the early steps of mini-F DNA replication, we examined the effects of RepE and the Escherichia coli proteins DnaA and HU on the localized melting of ori2 DNA in a purified in vitro system. We found that the binding of RepE to an iteron causes a 50^° bend at or around the site of binding. RepE and HU exhibited synergistic effects on the localized melting within the ori2 region, as detected by sensitivity to the single-strand specific P1 endonuclease. This opening of duplex DNA occurred around the 13mer of ori2, whose sequence closely resembles the set of 13mers found in the chromosomal origin oriC. Further addition of DnaA to the reaction mixture increased the efficiency of melting and appeared to extend melting to the adjacent AT-rich region. Moreover, DNA melting with appreciably higher efficiencies was observed with mutant forms of RepE that were previously shown to be hyperactive both in DNA binding in vitro and in initiator activity in vivo. We propose that the binding of RepE to four iterons of ori2 causes bending at the sites of RepE binding and, with the assistance of HU, induces a localized melting in the 13mer region. The addition of DnaA extends melting to the AT-rich region, which could then serve as the entry site for the DnaB-DnaC complex, much as has been documented for oriC- dependent replication.     

A Novel Class of Herbicides (Specific Inhibitors of Imidazoleglycerol Phosphate Dehydratase)

A new mode of herbicidal action was established by finding specific inhibitors of imidazoleglycerol phosphate dehydratase, an enzyme of histidine (His) biosynthesis. Three triazole phosphonates inhibited the reaction of the enzyme with Ki values of 40 [plus or minus] 6.5, 10 [plus or minus] 1.6, and 8.5 [plus or minus] 1.4 nM, respectively, and were highly cytotoxic to cultured plant cells. This effect was completely reversed by the addition of His, proving that the cytotoxicity was primarily caused by the inhibition of His biosynthesis. These inhibitors showed wide-spectrum, postemergent herbicidal activity at application rates ranging from 0.05 to 2 kg/ha.     

Application of bacterial magnetic particles as novel DNA carriers for ballistic transformation of a marine cyanobacterium

Summary Bacterial magnetite particles (BMPs) of 50 to 100nm diam were used as DNA carriers for the ballistic transformation of the marine cyanobacteriumSynechococcus. BMPs were bombarded into the cyanobacterial cells at several bombardment velocities using a particle gun. Successful transformation and gene expression were confirmed by Southern hybridization and CAT assay, respectively. The BMPs were also observed in the cyanobacterial cells by transmission electron microscopy. These results suggested that BMPs can be used as carriers for introducing DNA into bacterial cells.     

Photomicrobial sensors for selective determination of phosphate

A photomicrobial sensor consisting of immobilized Chlorella vulgaris and an oxygen electrode has been developed for selective determination of phosphate. When 40 mM phosphate was added to the sensor system, the photocurrent increased to a maximum under light irradiation with a response time of 1 min. The current increased with increasing phosphate concentration in the range 8–70 mM. Selectivity of the sensor was satisfactory. Good agreement was obtained between the phosphate concentrations in lake water determined by the photomicrobial sensor and by conventional colorimetry (correlation coefficient 0.96).     

Photomicrobial electrode for selective determination of sulphide

Summary A photomicrobial electrode, which uses the photosynthetic bacteria Chromatium sp. in conjunction with a hydrogen electrode, was developed for the determination of sulphide. The response time of the photomicrobial electrode was 5–10 min. A linear relationship was obtained between the current of the electrode and the sodium sulphide concentration below 3.5 mM. The minimum detectable concentration of sodium sulphide was 0.4 mM. Selectivity of the sensor is satisfactory. A good agreement was obtained between the photomicrobial electrode and the ethylene blue method (correlation coefficient: 0.90).     

A cold-labile acetyl-coenzyme-A hydrolase from the supernatant fraction of rat liver. Reactivation and reconstitution of the active species from the inactive monomer

An acetyl-coenzyme-A hydrolase from the supernatant fraction of rat liver is known to be rapidly inactivated at low temperature. Loss of catalytic activity is accompanied by apparent dissociation of tetrameric and dimeric forms of the enzyme into monomers. It was found that rewarming under appropriate conditions almost completely reversed the cold-induced inactivation and dissociation of the enzyme: At a protein concentration of 14 micrograms/ml, simple rewarming only partially restored the enzyme activity (less than 3% of the original activity), but at a higher concentration of the enzyme or in the presence of 1 mg/ml bovine serum albumin, the reactivation by warming was greater. Warming at 37 degrees C appeared to be optimal for reactivation; warming at 25 degrees C or at 43 degrees C was less effective. Longer exposure to cold did not affect reactivation on rewarming, but on repeated inactivation and reactivation the reactivation decreased to some extent, especially at lower concentrations of enzyme protein. Among various nucleotides tested, ATP greatly enhanced the restoration of the activity, while ITP, UTP and ADP were less effective and AMP, GTP, TTP and CTP had little effect. At an enzyme-protein concentration of 14 micrograms/ml, 2 mM ATP restored the enzyme activity to about 70% of that before cold treatment, while acetyl-CoA (0.5 mM) restored the activity about 50%. High concentrations of phosphate (0.92 M) and pyrophosphate (0.45 M) restored about 80% and 95%, respectively, of the original activity. Sucrose density gradient centrifugation of the active dimer at high enzyme concentration at 4 degrees C for 20 h produced a monomeric form without catalytic activity. Gel filtration showed that simple rewarming mostly converted the monomeric enzyme obtained in this way to the dimeric form, whereas on rewarming with ATP the monomer was mostly converted to a tetrameric form. The dimeric and tetrameric forms both had catalytic activity.     

Reexamination of paternal age effect in Down's syndrome

Summary Paternal age distribution for 1279 cases of Down's syndrome born in 1952–1968 was compared with the corresponding distribution for the general population, corrected for the maternal age as well as for the year of birth of the patients. Although there was no difference in the mean paternal age, the two distributions differed significantly, largely due to the excess of fathers aged 55 years and over and to the deficit of those aged 40–44 years in the patients born to mothers aged 30 years and over. The overall pattern of the relative incidence of Down's syndrome with advancing paternal age, with maternal age controlled, seems consistent with the hypothesis proposed by Stene et al. (1977). It increased from 0.8 for fathers aged 20–24 years slowly up to 1.2 for those aged 45–49 years, though with an intermediate drop to 0.8 at the age of 40–44 years, and then sharply to 2.4 for those aged 55 years and over. This rising pattern of the relative incidence with paternal age was essentially the same for the patients born in 1952–1960 and for those born in 1961–1968, although the slope was less steep in the latter than in the former group.This paper is dedicated to Professor Heinrich Schade in honor of his 70th birthday     

A specific microbial sensor for formic acid

Summary A microbial sensor consisting of immobilized Clostridium butyricum, two gas permeable Teflon membranes and fuel cell type electrode was suitable for the determination of formic acid. When the sensor was inserted into the sample solution containing formic acid, the current increases to a steady state with a response time of 20 min. The relationship between the steady state current and the formic acid concentration is linear up to 1 000 mg l^–1. The currents are reproducible with an average relative error of 5%. Selectivity of the sensor is satisfactory. Results obtained with this sensor and by gas chromatography were in good agreement (regression coefficient; 0.98) when the cultivation medium of Aeromonas formicans was employed. Immobilized Clostridium butyricum is stable for more than 20 days.