Destruction of tumor cells by monokines released from activated human blood monocytes: evidence for parallel and additive effects of IL-1 and TNF

Summary The incubation of human peripheral blood monocytes with endotoxins activates the cells to lyse tumorigenic targets directly and also induces the production and release into the culture medium of factors that produce lysis of mouse-transformed fibroblasts L-929 (tumor necrosis factor (TNF)-sensitive) and human A-375 melanoma cells (interleukin-1 (IL-1)- and TNF-sensitive). Immunoblotting analysis revealed that the culture medium of endotoxin-activated but not of control monocytes contained both IL-1 and TNF with a molecular weight of 17,000 daltons each. TNF activity was determined by lysis of L-929 cells, and IL-1 activity was measured by the proliferation of D-10 cells. The production of IL-1 and TNF was concentration-dependent, and the amounts of these monokines were paralleled. The antitumor activity of the culture supernates from endotoxin-treated monocytes was significantly decreased by incubation with heterologous antisera to IL-1, TNF, or both. Recombinant human IL-1 and TNF were used in parallel experiments and as positive controls. Each monokine used produced cytotoxic effects in susceptible targets. The combination of IL-1 and TNF, which more likely resembles culture supernates of activated macrophages, produced an additive antitumor cytotoxicity effect.     

Effect of ultraviolet irradiation of bacteriophage f1 DNA on its conversion to replicative form by extracts of Escherichia coli

Summary When DNA of phage X174 or phage f1 is used as a template after exposure to ultraviolet radiation, the conversion of single-stranded DNA to replicative form by cell-free extracts of Escherichia coli is inhibited. The extend of synthesis is proportional to the distance of a pyrimidine dimer from a specific origin of replication as calculated from the random location of dimers at various UV doses. The results therefore indicate that the initiation of DNA synthesis on these phage DNAs occurs normally at a specific site, and that chain elongation is blocked when replication reaches a photo product in the template. Reinitiation of DNA synthesis distal to the lesion does not occur.Part of this work was supported by a Grant-in-Aid for scientific research from the Ministry of Education, Science and Culture, Japan.     

Individualized Mutation Detection in Circulating Tumor DNA for Monitoring Colorectal Tumor Burden Using a Cancer-Associated Gene Sequencing Panel

Background

Circulating tumor DNA (ctDNA) carries information on tumor burden. However, the mutation spectrum is different among tumors. This study was designed to examine the utility of ctDNA for monitoring tumor burden based on an individual mutation profile.

Methodology

DNA was extracted from a total of 176 samples, including pre- and post-operational plasma, primary tumors, and peripheral blood mononuclear cells (PBMC), from 44 individuals with colorectal tumor who underwent curative resection of colorectal tumors, as well as nine healthy individuals. Using a panel of 50 cancer-associated genes, tumor-unique mutations were identified by comparing the single nucleotide variants (SNVs) from tumors and PBMCs with an Ion PGM sequencer. A group of the tumor-unique mutations from individual tumors were designated as individual marker mutations (MMs) to trace tumor burden by ctDNA using droplet digital PCR (ddPCR). From these experiments, three major objectives were assessed: (a) Tumor-unique mutations; (b) mutation spectrum of a tumor; and (c) changes in allele frequency of the MMs in ctDNA after curative resection of the tumor.

Results

A total of 128 gene point mutations were identified in 27 colorectal tumors. Twenty-six genes were mutated in at least 1 sample, while 14 genes were found to be mutated in only 1 sample, respectively. An average of 2.7 genes were mutated per tumor. Subsequently, 24 MMs were selected from SNVs for tumor burden monitoring. Among the MMs found by ddPCR with > 0.1% variant allele frequency in plasma DNA, 100% (8 out of 8) exhibited a decrease in post-operation ctDNA, whereas none of the 16 MMs found by ddPCR with < 0.1% variant allele frequency in plasma DNA showed a decrease.

Conclusions

This panel of 50 cancer-associated genes appeared to be sufficient to identify individual, tumor-unique, mutated ctDNA markers in cancer patients. The MMs showed the clinical utility in monitoring curatively-treated colorectal tumor burden if the allele frequency of MMs in plasma DNA is above 0.1%.     

Measurement of the Critical Nyctoperiod by Lemna paucicostata 6746 Grown in Continuous Light

The short-day duckweed Lemna paucicostata 6746 could be inducedto flower in two days at 26C when continuous illumination forentrainment was followed by continuous darkness. This 48-h darkperiod or the minimum darkness requirement for floral inductionwas called the induction period. The length of the inductionperiod (IP) was routinely computed as the number of 24-h cyclesusing the equation of regression of flower number in logarithmon culture time. A light pulse given about 7 h after the startof the induction period increased the apparent IP value fromtwo to three, suggesting that the interrupted first day hadfunctioned as a noninductive day. A pulse given at any otherpart of the induction period did not modify the IP value. Thelight-sensitive part is probably the inducible phase, and thefirst 7-h period of darkness terminated by it seems to be thecritical nyctoperiod. These and relevant facts suggest thatthe light-off oscillator measures the critical night length,7 h. Either red or far-red irradiation at the inducible phase extendedthe IP value by one. No red/far-red photoreversibility was detected.As expected, however, red or far-red irradiation of any otherpart of the critical nyctoperiod could not modify the IP value. (Received February 8, 1985; Accepted May 14, 1985)     

Involvement of murine β-1,4-galactosyltransferase V in lactosylceramide biosynthesis

Human β-1,4-galactosyltransferase (β-1,4-GalT) V was shown to be involved in the biosynthesis of N-glycans, O-glycans and lactosylceramide (Lac-Cer) by in vitro studies. To determine its substrate specificity, enzymatic activity and its products were analyzed using mouse embryonic fibroblast (MEF) cells from β-1,4-GalT V (B4galt5)-mutant mice. Analysis of expression levels of the β-1,4-GalT I-VI genes revealed that the expression of the β-1,4-GalT V gene in B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells are a half and null when compared to that of B4galt5 ( +/+ )-derived MEF cells without altering the expression levels of other β-1,4-GalT genes. These MEF cells showed no apparent difference in their growth. When β-1,4-GalT activities were determined towards GlcNAcβ-S-pNP, no significant difference in its specific activity was obtained among B4galt5 ( +/+ )-, B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells. No significant differences were obtained in structures and amounts of N-glycans and lectin bindings to membrane glycoproteins among B4galt5 ( +/+ )-, B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells. However, when cell homogenates were incubated with glucosylceramide in the presence of UDP-[(3)H]Gal, Lac-Cer synthase activity in B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells decreased to 41% and 11% of that of B4galt5 ( +/+ )-derived MEF cells. Consistent with this, amounts of Lac-Cer and its derivative GM3 in B4galt5 ( -/- ) -derived MEF cells decreased remarkably when compared with those of B4galt5 ( +/+ )-derived MEF cells. These results indicate that murine β-1,4-GalT V is involved in Lac-Cer biosynthesis.     

Gap formation and regeneration of tropical mangrove forests in Ranong,Thailand

Tropical mangrove forests are characterized by clear zonation along a tidal gradient, and it has been supposed that the zonation is primarily controlled by soil factors. However, effects of disturbance on mangrove forests are still not well understood and may play an important role on the vegetation patterns and forest dynamics in some forest formations. In this study, the pattern of disturbance regime and its effects on regeneration of tropical mangrove forests along a tidal gradient were investigated in Ranong, Thailand. We established one or two 0.5 ha plots in four vegetation zones, i.e. Sonneratia alba–Avicennia alba zone, Rhizophora apiculata zone, Ra – Bruguiera gymnorrhiza zone, Ceriops tagal–Xylocarpus spp. zone. Gap size (percentage gap area to total study area and individual gap size) was the largest in Sa–Aa zone which is located on the most seaward fringe, and it declined from seaward to inland. Canopy trees of S. alba and A. alba had stunted trunks and showed low tree density. On the contrary, canopy dominants in the other three inland zones, e.g. R. apiculata, B. gymnorrhiza, and Xylocarpus spp., had slender trunks and showed high tree density. Accordingly, differences in disturbance regime among the four zones were resulted from the forest structural features of each zone. Disturbance regime matched with regeneration strategies of canopy dominants. Seedlings and saplings of S. alba and A. alba, which need sunny condition for their growth, were abundant in gaps than in understorey. By contrast, R. apiculata, B. gymnorrhiza, and Xylocarpus spp., which can tolerate less light than S. alba and A. alba, had greater seedling and sapling density under closed canopy than gaps. Many large gaps may enhance the abundance of S. alba and A. alba in Sa–Aa zone, and a few small gaps may prevent the light demanding species to establish and grow in the other inland zones. Correspondence of disturbance regime and regeneration strategies (e.g. light requirement) of canopy dominants may contribute to the maintenance of the present species composition in each of the vegetation zones.     

Geographical Distribution of Adolescent Body Height with Respect to Effective Day Length in Japan: An Ecological Analysis

The height of Japanese youth raised in the northern region tends to be greater than that of youth raised in the southern region; therefore, a geographical gradient in youth body height exists. Although this gradient has existed for about 100 years, the reasons for it remain unclear. Consideration of the nutritional improvement, economic growth, and intense migration that has occurred in this period indicates that it is probably the result of environmental rather than nutritional or genetic factors. To identify possible environmental factors, ecological analysis of prefecture-level data on the body size of 8- to 17-year-old youth averaged over a 13-year period (1996 to 2008) and Japanese mesh climatic data on the climatic variables of temperature, solar radiation, and effective day length (duration of photoperiod exceeding the threshold of light intensity) was performed. The geographical distribution of the standardized height of Japanese adolescents was found to be inversely correlated to a great extent with the distribution of effective day length at a light intensity greater than 4000 lx. The results of multiple regression analysis of effective day length, temperature, and weight (as an index of food intake) indicated that a combination of effective day length and weight was statistically significant as predictors of height in early adolescence; however, only effective day length was statistically significant as a predictor of height in late adolescence. Day length may affect height by affecting the secretion of melatonin, a hormone that inhibits sexual and skeletal maturation, which in turn induces increases in height. By affecting melatonin production, regional differences in the duration of the photoperiod may lead to regional differences in height. Exposure to light intensity greater than 4000 lx appears to be the threshold at which light intensity begins to affect the melatonin secretion of humans who spend much of their time indoors.     

Rolling-Circle Plasmid pKYM Re-initiates DNA Replication

It is believed that rolling-circle plasmids are incapable ofre-initiation since they have to maintain their copy numberand this is one of the difierences between plasmids and phagesas phi-x174. To examine whether a rolling-circle plasmid pKYMis incapable of re-initiating DNA replication, we constructeda plasmid that carries both the pKYM origin (fragment 13, 173bp) and its truncated origin (fragment 32, 56 bp) in the sameorientation. This plasmid yielded two smaller plasmids in thepresence of RepK, an initiator protein. We showed that RepKcan bind to the fragment 13 but not to fragment 32 which lacksthe 3'-moiety of fragment 13. These results imply that RepKinitiates DNA replication from fragment 13 and terminates atfragment 32, then the same RepK is used for re-initiation ofreplication from the fragment 32 region. pKYM is likely to bea unique plasmid that re-initiates DNA replication like a phagephi-x174.