Modification of regression of virally xenogenized tumor cells by cyclophosphamide and busulfan

Summary Rat fibrosarcoma cells infected with Friend leukemia virus (FV-KMT-17) grow for a short time and then regress spontaneously in syngeneic hosts. This regression mechanism was examined by analyzing the immunomodulating action of the antitumor drugs busulfan (BU) and cyclophosphamide (CY). In preliminary experiments, the optimum dosages of BU and CY for the enhancement of DTH responses to SRBC were 10 mg/kg and 40 mg/kg respectively. Treatment of rats with BU (10 mg/kg) on day 5 induced the regression of KMT-17 cells, while in contrast, the same drug delayed the spontaneous regression of FV-KMT-17 cells. Pretreatment with CY (40 mg/kg) on day 5 did not affect the growth of KMT-17 or FV-KMT-17 cells. After the same treatment schedule, BU inhibited humoral antibody formation against SRBC and against virus-associated antigen (VAA), NK cell activity, and ADCC effector cell activity. On the other hand, CY did not affect the activities of NK cells or ADCC effector cells, although it significantly augmented the DTH responses to SRBC and the production of antibody to VAA but had no effect on production of antibodies to SRBC. These results suggest that NK cells and ADCC may play an important role in the initial stage of the spontaneous regression of FV-KMT-17 cells.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education Abbreviations used: BU, busulfan; CY, cyclophosphamide; PFC assay, plaque forming cell assay; VAA, virus-associated antigen; NK cell, natural killer cell; ADCC, antibody dependent cellular cytotoxicity; MuLV, murine leukemia virus; DTH, delayed type hypersensitivity; SRBC, sheep red blood cells; C.I., cytotoxic index; CRBC, chicken red blood cells; IL-1, interleukin 1; IL-2, interleukin 2; IFN, interferon     

Isolation of a cAMP-requiring mutant in Escherichia coli K-12: evidence of growth regulation via N-acetylglucosamine metabolism controlled by cAMP

Abstract An adenylate cyclase gene ( cya ) mutant was mutagenized and an adenosine 3,5-cyclic monophosphate (cAMP)-requiring mutant (KM8161) was obtained on Davis minimal medium containing glucose in the presence or absence of cAMP. KM8161 also required N -acetylglucosamine for its growth instead of cAMP. Furthermore, the mutant could use neither glucosamine nor N -acetylglucosamine as the carbon source. These results indicate that the cAMP-requiring property is due to multiple mutations of a few genes involved in amino sugar metabolism in addition to cya . By genetic analysis of KM8161, one gene, which was tentatively termed cidA and located near 2 min on the chromosomal map, proved to be defective. Reversion of cidA mutation in KM8161 resulted in recovery of not only the cAMP-requiring phenotype but also non-utilization of amino sugars. When both cAMP and N -acetylglucosamine or glucosamine were added to the culture medium for KM8161, only N -acetylglucosamine could be utilized as the carbon source. These studies s strongly suggest that the cidA or cya mutation in KM8161 causes deficiency in different stages of amino sugar metabolism and the regulatory circuit of growth by cAMP is mediated via control of N -acetylglucosamine metabolism.     

Surface Charge Density Estimation of Vigna mungo Protoplasts Using a Fluorescent Dye, 9-Aminoacridine

We showed that the surface charge density of protoplasts canbe estimated by the 9-aminoacridine method. The estimated surfacecharge density of the protoplasts isolated from elongating regionsof Vigna mungo root was – 39 ? 8 mC/m². The negative surfacecharge density increased when protoplasts were treated withglutaraldehyde or when EDTA was added to the protoplast suspensionmedium. These results support the validity of our estimationof the surface charge density of protoplasts by the 9-aminoacridinemethod. The concentration of amino groups at the surface ofthe protoplasts was estimated to be 34 mC/m². (Received June 19, 1987; Accepted April 11, 1988)     

Application of 39K NMR Spectroscopy to Potassium Transport in Mung Bean Root Tips

The intracellular K⁺ concentration and its change in mung bean[Vigna mungo (L.) Hepper] root tips were investigated non-invasivelywith ³⁹K nuclear magnetic resonance spectroscopy using a membraneimpermeable shift reagent, dysprosium (III) tripolyphosphate[Dy(PPP_i)^7–₂]. The K⁺ resonance was shifted to highermagnetic field in proportion to the concentration of the shiftreagent. In addition to a reference capillary peak for measuringthe K⁺ concentration, two well-resolved peaks (intra- and extracellularK⁺ resonances) were observed in the 39K NMR spectra of mungbean root tips. The intracellular K⁺ concentration was determinedto be 41 mM, which was similar to the value obtained by flamephotometry. When 20 mM KCl was added to the external medium,the intensity of the intracellular K⁺ resonance gradually increasedand the net K⁺ uptake rate was calculated to be 4.1 micromolesper gram fresh weight per hour. After removal of KCl from theperfusion medium, the intracellular K⁺ concentration considerablydecreased. With ³¹P NMR method, 2.5 mM Dy(PPP_j)^7–1₂ and20 mM KCl had little effect on the ATP level in the cells. Wehave indicated that the ³⁹K NMR method can be used to determinethe K⁺ levels and net fluxes of the K⁺ transport in perfusedroot tips successively. (Received April 6, 1988; Accepted September 29, 1988)     

Asymmetric Reduction of 4-Oxoisophorone Using Immobilized Thermophilic Bacteria Under Conditions of Controlled Cell Growth

Asymmetric reduction of 2,6,6,-trimethyl-2-cyclohexene-l,4-dione (4-oxoisophrone) to (6R)-2,2,6-trimethyl-1,4-cyclohexane-dione((3R)-dihydro-4-oxoisophorone) was catalysed by immobilized thermophilic bacteria, Thermomonospora curvata JTS 321. Because of leakage of entrapped cells from gel beads during reactions using culture medium, we optimized the medium to allow the microbial conversion under conditions of controlled cell growth. Of the media screened, liver infusion medium was found to be the most suitable and microbial conversion was achieved without cell leakage from the immobilized gels. Immobilized T. curvata cells were repeatedly used for the asymmetric reduction of 4-oxoisophorone, more than 15 times, with an extent of conversion of 50%.     

Participation of glutamic acid 23 of T4 endonuclease V in the beta-elimination reaction of an abasic site in a synthetic duplex DNA.

T4 endonuclease V catalyzes the hydrolysis of the glycosyl bond of a thymine dimer in a DNA duplex and the cleavage of the 3'-phosphate by beta-elimination. We have previously identified a catalytic site for the first reaction (pyrimidine dimer-glycosylase activity) by systematic mutagenesis (Doi et al. Proc. Natl. Acad. Sci. USA 1992 in press) and by x-ray crystallography (Morikawa et al. Science, 256: 523-526, 1992). The results showed that replacement of Glu23 with either glutamine or aspartic acid completely abolished the glycosylase activity. We describe the investigation of the second reaction (apurinic/apyrimidinic endonuclease activity), using twenty two mutants of T4 endonuclease V plus a DNA mini duplex containing an abasic site. Replacement of Glu23 by glutamine abolished the second reaction, but replacement with aspartic acid did not. The pH optima of the mutant (23 Asp) and the wild type were found to be 5.0 and 5.5, respectively. We conclude that the carboxylate anion in position 23 may act as a general base in the beta-elimination reaction of the endonuclease.     

Effects of temperature and neuroactive substances on hypothalamic neurones in vitro: possible implications for the induction of fever.

This paper reviews some of our findings which have shown the usefulness of in vitro methods in the study of hypothalamic neurones. (1) Membrane current analyses of dispersed neurones of the rat preoptic and anterior hypothalamus (POA) during thermal stimulation have revealed that warm-sensitive neurones are endowed with a non-inactivating Na+ channel having a high Q10 in the hyperthermic range (35-41 degrees C). (2) A brain slice study has shown that neurones in the organum vasculosum lamina terminalis (OVLT) region have much higher sensitivity to PGE2 than POA neurones. This provides further evidence of a critical role of the OVLT in translation of blood-borne cytokine signals into brain signals for fever induction. (3) Local application of IL-1 beta and IFN alpha altered the activity of thermosensitive (TS) neurones and glucose responsive (GR) neurones in vitro in an appropriate way to produce fever and anorexia. While the responses to IL-1 beta required the local release of prostaglandins, the responses to IFN alpha were found to be mediated by opioid receptor mechanisms. (4) The responses of POA TS neurones and VMH GR neurones to IL-1 beta but not those to IFN alpha, were reversibly blocked by alpha MSH, an endogenous antipyretic peptide. Thus, immune cytokines and their related neuroactive substances may affect hypothalamic TS and GR neurones thereby producing elaborately regulated changes in homeostatic functions such as thermoregulation (fever) and feeding (anorexia), which are considered as host defence responses.