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     

Selection of microorganisms which produce raw-starch degrading enzymes

Summary Microorganisms which produce strong raw-starch degrading enzymes were isolated from soil using a medium containing a unique carbon source, -amylase resistant starch (-RS), which is insoluble in water and hardly digested with Bacillus amyloliquefaciens -amylase. Among the isolates, three strains showing high activities were characterized. Two of them, K-27 (fungus) and K-28 (yeast), produced -amylase and glucoamylase, and the final product from starch was only glucose. The third strain, K-2, was a bacterium and produced -amylase, which produced glucose and malto-oligosaccharides from starch. The enzyme preparation of these strains degraded raw corn starch rapidly.     

Bromodeoxyuridine-immunohistochemistry on cellular differentiation and migration in the fundic gland of Xenopus laevis during development

Summary Cellular differentiation and migration in the fundic glands of adult and larval Xenopus laevis have been examined using bromodeoxyuridine-immunohistochemistry. In the adult fundic gland, cumulative labeling with bromodeoxyuridine revealed a proliferative cell zone between the surface mucous cells and mucous neck cells, in what is referred to as the neck portion of the gland. The labeling-index of mucous neck cells had rapidly increased by week-5. The labeling-index of oxynticopeptic cells showed a more delayed increase until week-7, coincident with the decrease in the labeling of mucous neck cells. In the immature fundic glands of larvae, the labeled proliferating cells were randomly distributed throughout the developing gastric mucosa. During metamorphosis, the labeling-index of immature epithelial cells was highest at stage 63. Following administration of bromodeoxyurdine at this, stage, there was no significant loss of labeled epithelial cells during the metamorphosing period. Furthermore, there was no significant difference in the labeling-indices among the epithelial cells, such as surface mucous cells/generative cells, mucous neck cells, and oxynticopeptic cells, 7 days after administration. Cellular differentiation and migration pathways of epithelial cells in the fundic gland of adult X. laevis and its larvae are discussed.     

Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli

Summary DNA fragments of 3.4 kb containing the gyrB gene were cloned from Escherichia coli KL-16 and from spontaneous nalidixic acid-resistant mutants. The mutations (nal-24 and nal-31) had been determined to be in the gyrB gene by transduction analysis. Nucleotide sequence analysis of the cloned DNA fragments revealed that nal-24 was a G to A transition at the first base of the 426th codon of the gyrB gene, resulting in an amino acid change from aspartic acid to asparagine, and nal-31 was an A to G transition at the first base of the 447th codon, resulting in an amino acid change from lysine to glutamic acid. This indicates that mutations in the gyrB gene are responsible for nalidixic acid resistance.     

Differentiation of Photorespiratory Activity between Mesophyll and Bundle Sheath Cells of C4 Plants II. Peroxisomes of Panicum miliaceum L.

Peroxisomes were isolated by sucrose density gradient centrifugationfrom mesophyll and bundle sheath protoplasts of a C₄ plant,Panicum miliaceum L. The equilibrium density in the gradientwas 1.25 for bundle sheath peroxisomes and 1.23 for mesophyllperoxisomes, the former density being similar to that of peroxisomesof wheat mesophyll protoplasts. Photorespiratory and other microbody enzymes were assayed forthe peroxisomes of P. miliaceum to detect possible differentiationat an enzyme level. The specific activities of photorespiratoryenzymes, except for hydroxypyruvate reductase, in bundle sheathperoxisomes were 40–60% of those in wheat peroxisomes,when compared on a protein basis, and only 20–30% in mesophyllperoxisomes. However, peroxisomes from both cell types containedsignificant levels of all the enzymes involved in the photorespiratoryglycolate pathway, when compared with castor bean glyoxysomes.The activity of hydroxypyruvate reductase in the peroxisomesof P. miliaceum was comparable to or higher than that in wheatperoxisomes. Two ß-oxidation enzymes and urate oxidasewere detected in the peroxisomes in a similar level to thatin wheat peroxisomes. These results suggest that the peroxisomes of mesophyll andbundle sheath cells of P. miliaceum are essentially similarto those of C₃ plants, and that they cannot be differentiatedexcept for a difference in equilibrium density in a sucrosegradient. (Received December 24, 1984; Accepted April 9, 1985)     

Two-dimensional gel studies of genetic variation in the plasma proteins of Amerindians and Japanese

Summary Genetic variation has been studied in plasma samples from 107 Amerindian children and their parents, and 110 Japanese children and their parents by means of two-dimensional polyacrylamide gel electrophoresis. Twenty-three polypeptides were scored; the identity of nine of these is at present still unknown. Genetic variation was encountered in 11 of these polypeptides. We have previously reported that the index of heterozygosity was 6.2±0.7% for 20 randomly selected, silver stained polypeptides scored for genetic variation in Caucasoids (Rosenblum et al. 1983b). For technical reasons only 11 of these 20 polypeptides could be routinely scored in preparations from the Amerindian samples. For these 11 polypeptides, the indices of heterozygosity in the three populations were: Amerindians, 4.5±0.6%; Japanese, 5.7±0.7%; Caucasoids, 8.0±1.1%. Even with these relatively small numbers some striking ethnic differences as regards individual polypeptides are apparent.     

Changes of aldolase A and B messenger RNA levels in rat liver during azo-dye-induced hepatocarcinogenesis

The expression of aldolase A and B mRNAs during azo-dye-induced carcinogenesis in rat liver was examined. After feeding the dye for 18 weeks, the level of aldolase A mRNA increased to about 11 times that in a normal liver, with the concomitant decrease of aldolase B mRNA level to about 25% of that in a normal liver. These changes did not occur progressively during the carcinogenesis, but occurred as an additional phase after 4 week-feeding of the azo-dye. At this stage, the levels of aldolase A and B mRNAs were about 7 times and 45% of that in a normal liver, respectively. This biphasic pattern in the aldolase isozyme expression in the azo-dye-fed rat liver is discussed together with the kinetic data of the enzyme activity.     

Overlapping of the coding regions for alpha and gamma components of penicillin-binding protein 1 b in Escherichia coli

Summary The mode of biosynthesis of penicillin-binding protein(PBP)-1 b in Escherichia coli was investigated by use of the plasmid carrying the ponB(PBP-1 b) gene region. Analyses of the products synthesized in minicells and in vitro showed that PBP-1 b was synthesized as two molecular species corresponding to the and components of PBP-1 b. The coding regions for the and components were located within the ca. 3.7 kb MluI-HincII fragment and transcribed in the direction from the HincII to the MluI site. The capacity for producing the component was abolished by a deletion extending to the MluI site ca. 0.7 kb inward from the HincII end of the ca. 3.7 kb fragment; the remaining 3.0 kb region with the MluI site at both ends directed the production of the component alone. The production of the component was enough to correct all the known defects caused by a ponB mutation. In addition to these results, the analyses for cross-reacting materials produced in correspondence to the various deletions indicated that the coding regions for the and components overlapped and that the N-terminal portion was responsible for the difference between the two components. The distal region about 0.7 kb long inward from the MluI end of the MluI-HincII fragment was dispensable for producing the functional PBP-1 b, although the PBP-1 b produced was curtailed. By a larger distal deletion reaching almost to the middle of the MluI-HincII fragment, the polypeptide produced for PBP-1 b lost the ability to bind penicillin and still retained a low but significant activity for glycan synthesis. We suggest, therefore, that the polypeptide portion required for transglycosylase activity resides on the N-terminal half of PBP-1 b, followed by the middle portion necessary for penicillin-binding and the C-terminal part dispensable for the function of PBP-1 b.     

A new approach to high sensitivity differential hybridization

We describe a new approach to differential hybridization, designed to identify cDNA clones representing rare mRNA species. Duplicate filters carrying a library of cDNA from phorbolmyristate acetate (PMA)-induced EL-4 cells in λgt11 were hybridized with high concentrations of unlabeled, cloned, single-stranded cDNA from induced and control EL-4 cells, respectively. Plaques binding single-stranded cDNA were revealed by a second round of hybridization with ³⁵S-labeled DNA complementary to the vector moiety of the single-stranded cDNA. Plaques corresponding to PMA-induced mRNAs occurring at a level of about 1 part in 15000 were isolated. We believe the method is at least ten times more sensitive than conventional differential hybridization.