Early Lag Phase Research Articles

Abstract 2939: Harnessing in vivo CRISPR screen to uncover metabolic dependencies during cancer progression

Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with the worst prognosis and limited treatment options. TNBC cells undergo metabolic reprogramming in response to nutrient limitations and changes in the tumor microenvironment throughout cancer progression. In the early stages, tumor cells are found to prioritize nutrient uptake and biosynthesis, but as they grow, intensified competition for nutrients fosters the emergence of subpopulations of cancer cells that excel in utilizing specific nutrients. The substantial metabolic pathways and genes that regulate each phase of cancer progression remain poorly understood. To address this, we have developed a stable integration, site-specific, and inducible CRISPR-Cas9 system that allows for temporal control of genetic perturbation. We conducted an unbiased in vivo loss-of-function CRISPR screen aimed at identifying metabolic vulnerabilities at different phases of cancer progression of TNBC. The inducible CRISPR system was activated only at the desired study period of each phase, thus capable of uncovering metabolic dependencies for TNBC proliferation at each distinct phase of cancer progression. As many large-scale CRISPR screens have previously been performed to identify gene dependencies for cancer progression in vitro, our CRISPR screen could address the shortcomings of in vitro screens by identifying novel essential metabolic genes that are significantly important for cancer growth in vivo. Preliminary data from our CRISPR screen revealed different pathways to be significantly important for tumor growth and proliferation at different phases of TNBC progression. For example, we find that ferroptosis is altered during the early lag phase of TNBC cancer progression, whereas, at the later stage of cancer progression, metastatic tumors upregulate serine metabolism, suggesting that secondary tumors could have distinct metabolism from the primary sites. Altogether, the findings from this study could provide valuable insights into stepwise metabolic dependencies arising from microenvironmental limitations and identify potential therapeutic targets at each phase of cancer progression. Moreover, our CRISPR-Cas9 model holds promise for dissecting the complex mechanisms of other cancer progressions. Citation Format: Seow Qi Ng, Zhengwei Wu, Jessica A. Lidster, Wai Leong Tam. Harnessing in vivo CRISPR screen to uncover metabolic dependencies during cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2939.

Aggregation Dynamics Characteristics of Seven Different Aβ Oligomeric Isoforms-Dependence on the Interfacial Interaction.

The aggregation of β-amyloid (Aβ) peptides has been confirmed to be associated with the onset of Alzheimer's disease (AD). Among the three phases of Aβ aggregation, the lag phase has been considered to be the best time for early Aβ pathological deposition clinical intervention and prevention for potential patients with normal cognition. Aβ peptide exists in various lengths in vivo, and Aβ oligomer in the early lag phase is neurotoxic but polymorphous and metastable, depending on Aβ length (isoform), molecular weight, and specific phase, and therefore hardly characterized experimentally. To cope with the problem, molecular dynamics simulation was used to investigate the aggregation process of five monomers for each of the seven common Aβ isoforms during the lag phase. Results showed that Aβ(1-40) and Aβ(1-38) monomers aggregated faster than their truncated analogues Aβ(4-40) and Aβ(4-38), respectively. However, the aggregation rate of Aβ(1-42) was slower than that of its truncated analogues Aβ(4-42) rather than that of Aβpe(3-42). More importantly, Aβ(1-38) is first predicted as more likely to form stable hexamer than the remaining five Aβ isoforms, as Aβ(1-42) does. It is hydrophobic interaction mainly (>50%) from the interfacial β1 and β2 regions of two reactants, pentamer and monomer, aggregated by Aβ(1-38)/Aβ(1-42) rather than by other Aβ isoforms, that drives the hexamer stably as a result of the formation of the effective hydrophobic collapse. This paper provides new insights into the aggregation characteristics of Aβ with different lengths and the conditions necessary for Aβ to form oligomers with a high molecular weight in the early lag phase, revealing the dependence of Aβ hexamer formation on the specific interfacial interaction.

Microbial protein production from CO2, H2, and recycled nitrogen: Focusing on ammonia toxicity and nitrogen sources

Autotrophic hydrogen-oxidizing bacteria (HOB) have received increasing attention as a promising source of microbial protein (MP). However, most of the studies were conducted at relatively low ammonium concentrations (∼1 g N–NH 4 + /L). This study investigated the effect of high concentrations of ammonium and various nitrogen forms on the growth of Cupriavidus necator 335 for MP production. The cell growth was slightly inhibited at 2 g N–NH 4 + /L and was completely suppressed at 4 g N–NH 4 + /L. Inoculum from the early stage of exponential phase adapted better to the inhibition. Urea was the most preferred nitrogen source followed by nitrate and ammonium. Neither the ammonium concentration (within 2 g N/L) nor the different nitrogen sources altered the amino acid profiles significantly. This study demonstrated that waste streams containing high ammonium concentration (e.g., digestate) or with different forms of nitrogen (e.g., urine) could be used as a nitrogen source for MP production and thereby broadening the feedstocks and application of the process. • Ammonium above 2 g/L inhibited the growth of Cupriavidus necator 335. • C. necator 335 grew faster when subcultured at early lag-phase under 2 g N–NH 4 + /L. • Urea was the most preferred followed by nitrate and ammonium at 2 g N/L. • Ammonium at 0.26–2 g N/L did not affect amino acid profile significantly. • Different nitrogen sources did not affect amino acid profile significantly.

Histologic changes in early colonic anastomotic healing using autologous platelet-rich fibrin matrix

Anastomotic leakage represents a devastating post-surgical event with surgeon- and patient-related causes. The early lag phase of healing (day 0–4) is a time of increased vulnerability. The current role of tissue sealants as anastomotic adjuncts remains unclear, with controversial results reported. Platelet-rich plasma provides a reliable source of growth factors supporting wound healing. We aimed to assess the impact of a novel autologous platelet-rich fibrin (PRF) agent (Obsidian-ASG® matrix; GergMed Medizintechnik, Baiern, Germany) on colonic anastomotic healing in a porcine model. Left-sided colonic anastomoses were constructed in 16 pigs with reinforcement using the Obsidian-ASG® matrix in 12 animals. Animals were sacrificed on days 0, 4, 10 and 30, with histologic assessment of the anastomotic line along with evaluation of fibrotic tissue maturation, foreign body reactivity and mucin production. Collagen maturity and immunohistochemical changes of angiogenesis and infiltrating macrophage profiles were determined as well. There was no observable effect in matrix-treated animals on epithelial line thickness, granulation tissue formation, foreign body reactivity or mucosal restitution. Matrix-treated animals showed higher mucin production on day 4 accompanied by a more mature collagen and a greater degree of early angiogenic response with a higher infiltration density of M2 macrophages. Additionally, anastomotic burst pressure in matrix-treated animals was higher on day 0, 4, 10 and 30 (100, 100, 210, 160 vs. 40, 30, 60, 20 mm Hg, respectively). Obsidian-ASG® matrix may have beneficial effects on anastomotic healing by shifting the inflammatory response towards a mature and more stable collagen phenotype in the early phase.

H2S‐Induced Structural Changes of Insulin Protein in Early Lag Phase: Key to Detaining Amyloid Formation

Amyloid fibrils are formed by soluble proteins that assemble into insoluble fibers associated to many types of diseases such as Diabetes Type 2, and Alzheimer's Disease. Amyloid formation can be divided into three phases: the lag phase, where native protein unfolds and begins to self‐aggregate; the elongation phase, in which these aggregates grow into fibrils, and the steady phase, where saturation of mature fibrils occurs. Hen egg white lysozyme (HEWL) amyloid fibrils are inhibited in the presence of hydrogen sulfide (H2S) (Rosario, 2014). To further explain the means by which H2S inhibits amyloid fibrillation, insulin from bovine pancreas (IBP), was tested upon experimentation with increasing concentrations of H2S. ThT Fluorescence analysis revealed a decrease in peak intensity as the concentration of H2S increased in each sample, proving H2S to be an inhibitor of insulin amyloids. Atomic Force Microscopy (AFM) of the control sample showed an array of fibrillary structures. However, as the concentration of H2S increased, samples went from having a combination of fibrillary structures and small conglomerated spherules to the sole appearance of small spherules. Far UV‐Circular Dichroism spectra of the native protein and the protein interacting with H2S were very similar. However, non‐resonance Raman spectra revealed the presence of newly formed trisulfide bridges in the interaction between insulin and the H2S species as opposed to the disulfide bridges found in native and fibrillated insulin protein. The data suggest H2S‐protein interactions favor a structural change in the process of protein denaturing. The H2S species intervenes with amyloid formation early on during the lag phase inhibiting the formation of protofibrils and potential mature fibrils.Support or Funding InformationResearch reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103475. It was also supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number T34GM008419. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Strand specific RNA-sequencing and membrane lipid profiling reveals growth phase-dependent cold stress response mechanisms in Listeria monocytogenes.

The human pathogen Listeria monocytogenes continues to pose a challenge in the food industry, where it is known to contaminate ready-to-eat foods and grow during refrigerated storage. Increased knowledge of the cold-stress response of this pathogen will enhance the ability to control it in the food-supply-chain. This study utilized strand-specific RNA sequencing and whole cell fatty acid (FA) profiling to characterize the bacterium’s cold stress response. RNA and FAs were extracted from a cold-tolerant strain at five time points between early lag phase and late stationary-phase, both at 4°C and 20°C. Overall, more genes (1.3×) were suppressed than induced at 4°C. Late stationary-phase cells exhibited the greatest number (n = 1,431) and magnitude (>1,000-fold) of differentially expressed genes (>2-fold, p<0.05) in response to cold. A core set of 22 genes was upregulated at all growth phases, including nine genes required for branched-chain fatty acid (BCFA) synthesis, the osmolyte transporter genes opuCBCD, and the internalin A and D genes. Genes suppressed at 4°C were largely associated with cobalamin (B12) biosynthesis or the production/export of cell wall components. Antisense transcription accounted for up to 1.6% of total mapped reads with higher levels (2.5×) observed at 4°C than 20°C. The greatest number of upregulated antisense transcripts at 4°C occurred in early lag phase, however, at both temperatures, antisense expression levels were highest in late stationary-phase cells. Cold-induced FA membrane changes included a 15% increase in the proportion of BCFAs and a 15% transient increase in unsaturated FAs between lag and exponential phase. These increases probably reduced the membrane phase transition temperature until optimal levels of BCFAs could be produced. Collectively, this research provides new information regarding cold-induced membrane composition changes in L. monocytogenes, the growth-phase dependency of its cold-stress regulon, and the active roles of antisense transcripts in regulating its cold stress response.

Alzheimer peptides aggregate into transient nanoglobules that nucleate fibrils.

Protein/peptide oligomerization, cross-β strand fibrillation, and amyloid deposition play a critical role in many diseases, but despite extensive biophysical characterization, the structural and dynamic details of oligomerization and fibrillation of amyloidic peptides/proteins remain to be fully clarified. Here, we simultaneously monitored the atomic, molecular, and mesoscopic states of aggregating Alzheimer's amyloid β (Aβ) peptides over time, using a slow aggregation protocol and a fast aggregation protocol, and determined the cytotoxicity of the intermediate states. We show that in the early stage of fast fibrillation (the lag phase) the Aβ peptides coalesced into apparently unstructured globules (15-200 nm in diameter), which slowly grew larger. Then a sharp transition occurred, characterized by the first appearance of single fibrillar structures of approximately ≥100 nm. These fibrils emerged from the globules. Simultaneously, an increase was observed for the cross-β strand conformation that is characteristic of the fibrils that constitute mature amyloid. The number and size of single fibrils rapidly increased. Eventually, the fibrils coalesced into mature amyloid. Samples from the early lag phase of slow fibrillation conditions were especially toxic to cells, and this toxicity sharply decreased when fibrils formed and matured into amyloid. Our results suggest that the formation of fibrils may protect cells by reducing the toxic structures that appear in the early lag phase of fibrillation.

Accumulation of Biomass and Compositional Change Over the Growth Season for Six Photoperiod Sorghum Lines

Biomass sorghums [Sorghum bicolor (L.) Moench] are short-day photoperiod sensitive (PS) types, meaning that the crop will grow vegetatively late into the fall season in subtropical and temperate environments. This feature results in high biomass yield potential and mitigates drought susceptibility. The objective of this study is to assess biomass growth patterns and associated changes in composition over a growing season for PS sorghum. The experiment had a split-plot design with two replications, six PS sorghum genotypes, and 13 harvest dates. Harvest started at 60 days after planting (DAP) and continued every 15 days thereafter in both College Station (CS) and Corpus Christi (CC) in Texas, 2010. At each harvest, dry biomass yield, plant height and biomass composition (percent lignin and cellulose) were measured. For all genotypes, biomass accumulation followed a standard growth pattern which included an early lag phase, followed by a log phase of growth and finally, a general reduction in the rate of accumulation. The early lag phase ended at approximately 70 DAP, the log phase of growth ended at approximately 125 DAP, and biomass yields maximized between 180 and 225 DAP. The highest yielding genotype produced 24 Mg ha−1. Plant heights up to 400 cm were also measured between 180 and 225 DAP. Plant height and biomass yield patterns were similar, indicating that height is important to increase yield. Lignin and cellulose concentrations increased with time; at the highest yields (between 180 and 225 DAP), maximum lignin content were 14.5 to 15.5 % and maximum cellulose content was 31 to 32 %. As with yield potential, significant differences were detected for composition as well. The growth curves indicate that PS biomass sorghum yields sufficiently and can be harvested as early as 130 DAP with maximum sorghum biomass accumulation occurring between 180 and 225 days. Thus, with careful selection and deployment of biomass sorghum hybrids, the harvest season of biomass sorghum can be extended over a 3-month period in southern regions of the US

Optimization and control in bacterial Lag phase

The lag phase of bacterial growth is important from a medical and food safety perspective, but difficult to study due to the low density and metabolic rate of cells. A new study by Alon and colleagues reveals that the gene expression program during early lag phase prioritizes carbon source utilization enzymes over genes responsible for biomass accumulation. This cellular strategy ultimately maximizes growth, making the best long-term use of the new nutrient-rich environment.See research article: http://www.biomedcentral.com/1752-0509/7/136

Formation of low-dimensional crystalline nucleus region during insulin amyloidogenesis process

Insulin, as other amyloid proteins, can form amyloid fibrils at certain conditions. The self-assembled aggregation process of insulin can result in a variety of conformations, starting from small oligomers, going through various types of protofibrils, and finishing with bundles of fibrils. One of the most common consensuses among the various self-assembly processes that are suggested in the literature is the formation of an early stage nucleus conformation. Here we present an additional insight for the self-assembly process of insulin. We show that at the early lag phase of the process (prior to fibril formation) the insulin monomers self-assemble into ordered nanostructures. The most notable feature of this early self-assembly process is the formation of nanocrystalline nucleus regions with a strongly bound electron–hole confinement, which also change the secondary structure of the protein. Each step in the self-assembly process is characterized by an optical spectroscopic signature, and possesses a narrow size distribution. By following the spectroscopic signature we can measure the potency of amyloid fibrils inhibitors already at the lag phase. We further demonstrate it by the use of epigallocatechin gallate, a known inhibitor for insulin fibrils. The findings can result in a spectroscopic-based application for the analysis of amyloid fibrils inhibitors.

Phospholipase C and D in the commitment to survival or death in the early lag phase of tetrahymena cultures.

We made three kinds of experiments in order to elucidate aspects of physiological mechanisms involved in a series of specific events leading to either cell death or survival in the lag phase of culture growth. We studied the fate of newly inoculated Tetrahymena cells in small droplets at 'high' (more than 1000 cells ml(-1)) and 'low' cell densities (less than 600 cells ml(-1)) in a nutrionally complete, synthetic nutrient medium. Confirming previous results we found that the cells in high-density cultures multiplied to final densities around 500,000 cells ml(-1) and that cells in low-density cultures died before multiplying. The appearance of the cells was recorded with a video camera at 20 frames per second for 6 h or until they died. The results indicated that the death process took place within milliseconds. We also studied the effects of U 73122, an inhibitor of the phosphatidylinositol-specific phospholipase C, on cell survival at low densities. At low inhibitor concentrations low-density cells were rescued from dying. At high inhibitor concentrations all cells died, and phosphatidylinositol - but not phosphatidylserine and phosphatidylcholine - saved them. The results indicate that the paths leading to either cell death or to cell proliferation separate within the first few minutes after subcultivation into a new medium, since the first cells in each culture died within 4-30 min after inoculation. Our results also indicate that some PLC activity was required for stimulation of phospholipase D, and that cell death during the early lag phase is caused by a shortage in phosphatidylinositol before the phospholipase D activity is upregulated. These experiments are shedding light on the lethal consequences of a cellular depletion of the important signalling compound phosphatidylinositol in an in vivo system, and may help to elucidate mechanisms behind the century-old fact that eukaryote cells die when inoculated at too low a cell density to survive.

Phospholipase C and D in the commitment to survival or death in the early lag phase of Tetrahymena cultures

Phospholipase C and D in the commitment to survival or death in the early lag phase of Tetrahymena cultures

Effect of Antibiotic Plant Resistance on the Reproductive Fitness of the Russian Wheat Aphid (Homoptera: Aphididae)

Laboratory experiments were conducted to examine the effect of antibiosis on the reproductive fitness of the Russian wheat aphid, Diuraphis noxia (Mordvilko ). Cumulative nymphal production was modeled for 6 wheat, Triticum aestivum L., plant introductions (PI 47545, PI 94355, PI 94365, PI 151918, PI 225245, PI 225262) and a susceptible standard, ‘Stephens’ wheat, with a nonlinear regression procedure that used the logistic equation. It was not possible to model the resistant standard, ‘Border’ oat, because of early mortality and lack of nymphipositional activity. The maximum nymphal production (M) ranged from 68.3 (PI 225262) to 87.2 nymphs per female (Stephens wheat). The rate of increase, or increase in nymphal production rate, ranged from 2.4 (PI 225262) to 3.0 (Stephens wheat). The lag time ranged from 2.5 (Stephens wheat) to 2.7 log (days) (PI 151918). Lag time represented the period before the adult female began to produce nymphs. The original time scale (days), was transformed (natural log) to evaluate the early lag phase in these data. Contrasts of parameters M and K , and joint hypothesis testing (comparison of entire curve) among Stephens wheat and the selected resistant plant introduction lines indicated that there were significant differences between Stephens wheat and all plant introduction lines examined for all parameter comparisons ( M, K , and whole curve). Estimated models from trial 1 were applied to data from trial 2 to validate the suitability of the models to describe the nymphal production process. Examination of residuals (defined as the difference between the predicted value and the observed value) suggested that although the models tended to slightly overestimate nymphal production, they adequately represented nymphal production data for all plant introduction lines as well as Stephens wheat.

The agronomic effectiveness of reactive phosphate rocks 4. Early season lag in herbage production when reactive phosphate rock is used as a pasture fertiliser

Summary. The agronomic effectiveness of highly reactive North Carolina phosphate rock (NCPR), relative to triple superphosphate (TSP), was determined for individual harvests at 26 permanent pasture sites in the National Reactive Phosphate Rock Project from 1992 to 1995. The aim was to determine whether the performance of NCPR relative to TSP was consistent over the growing season. Poor performance of NCPR early in the growing season (early seasonal lags), and its subsequent improvement later in the season, was observed at 15 of the 26 sites in this project and for 20 of the 103 possible site x year combinations. At 3 additional sites the apparent early season lag in NCPR performance was followed by harvests where there was no phosphorus (P) response. At one P-leaching site with a long growing season the relative yields from NCPR plots increased as the year progressed and exceeded those from TSP late in th e season. The duration of the early season lag phase was associated with the rate of build-up in plant-available P over the 4-year duration of the trials. The lag disappeared after 1 or 2 years at sites with low P-sorbing, sandy soils where there was a significant build-up in Colwell P, but persisted for 4 years on high P-sorbing soils where limited increases in Colwell P occurred. The average early season yield reduction associated with use of NCPR, compared with using TSP, was 28%. Colwell soil test values alone did not provide a good indication of the likelihood of a seasonal effect occurring. The occurrence of an early season lag phase needs to be taken into account by farmers considering the use of RPR as reductions in feed supply at this time of the year have the potential to reduce stocking rates and farm profitability. The use of partially acidulated NCPR, or large applications of NCPR applied in the first year, were management strategies that minimised the occurrence of these seasonal effects. The suggestion that the early season lag effect is due to the inability of NCPR to meet P demands from newly establishing pasture legumes, and increased P requirements by pasture plants in cold conditions is also discussed.

Rate of tendon gap closure in an in vitro collagen gel matrix

Tendon gap closure was examined in a tissue culture model and found to have a similar time course as skin wound closure. Foot tendons from White Rock chickens were mounted in a collagen gel matrix and maintained with the use of Dulbecco's modified Eagle medium, containing fetal calf serum and antibiotics, for 4 weeks at 37 degrees C in an incubator. Gap distances between tendons were measured every 1 to 3 days and plotted against time as the contraction curve. After an initial lag period of 4 to 8 days, gap distance showed a progressive decrease. Gap closure rate was defined as the slope of the contraction curve, and it was found to be a function of initial gap distance (r = 0.643, p 0.045). The time necessary to reduce the initial gap distance by half had a significant correlation with the initial gap distance (r = 0.986, p < 0.001). Fibroblast migration began on days 2 to 3 after a 1- to 2-day lag period. Fibroblasts were visible in the tendon gap region before the start of collagen gel contraction. At this time, the fibroblast migration rate was 0.33 mm/day. A critical density of fibroblasts was necessary to start collagen gel contraction. Once the gap distance began to diminish, fibroblast migration measurements were hampered because the measurable area was decreasing. Collagen gel contraction reduced the measurable fibroblast migration rate by nearly half to 0.18 mm/day. A linear correlation was found between fibroblast distance traveled and time in culture during both the gel lag and gel contraction time periods. This tendon culture model may be potentially useful for wound healing studies because it allows for studies of fibroblast activity in the early lag phase when the cells populate the collagen lattice but before contraction of the gel occurs.

Regulation of the signal transduction program by drugs

The purpose of this paper was to clarify critical aspects of the behavior of signal transduction activity in normal and cancer cells. 1. 1. Signal transduction activity in the conversion of phosphatidylinositol through PI and PIP kinases and PLC to IP 3 is regulated at multiple sites. In liver, hepatomas and human carcinomas PIP kinase is the rate limiting enzyme and PLC activity is present in great excess. 2. 2. The steady-state signal transduction activity as measured by the three enzyme activities and IP 3 concentration was markedly up-regulated in rat hepatomas of different growth rates. The steady-state specific activities of the three signal transduction enzymes were elevated in ovarian carcinomas as compared to normal ovary. Increased enzyme activities were also observed in human breast carcinoma cells as compared to normal human breast parenchymal cells. In breast, ovarian and rat hepatoma cells as they go through lag, log and plateau phases, IP 3 concentration in the early lag phase increased 4.5- to 20-fold and PI and PIP kinase activities peaked in mid-log phase. These events returned to baseline levels in the plateau phase. PLC activity did not change. 3. 3. The bone marrow PI and PIP kinase activities in 3-day starvation were decreased to 13% and IP 3 concentration was reduced to 24%; at 1-day refeeding they returned to normal. PLC activity changed little. These alterations are in line with the rapid t 1 2 degradation rates (12 min) of PI and PIP kinases observed in studies with cycloheximide. By contrast, PLC has a long half-life. 4. 4. The molecular action of tiazofurin entails inhibition of IMP DH activity, decrease in GTP and IP 3 concentrations, reduction of ras and myc oncogene expression, and signal transduction enzyme activities. These events are followed by induced differentiation and apoptosis. There are also decreases in enzyme activities which have rapid turnover, including TdR kinase, dTMP synthase, and GPRT. In vitro studies indicated that these events are abrogated by addition of guanine which restores GTP concentration. Therefore, most or all these events were brought about by the reduced GTP concentration in the tiazofurin target cells. 5. 5. Quercetin and genistein are able to inhibit PI and PIP kinase activities and reduce IP 3 concentration in vivo and in tissue culture systems. These flavonoids are also inhibitors of cell proliferation and clonogenic ability in rat hepatoma 3924A and in human OVCAR-5 and MDA-MB-435 cells. Quercetin down-regulated the expression of c- myc and Ki- ras oncogenes and led to induced differentiation and apoptosis in K562 cells. Genistein reduced IP 3 concentration in vivo and in the tissue culture system. Genistein is anti-proliferative and has cytototoxicity in human carcinoma cells. All three drugs, tiazofurin, quercetin and genistein, act, in part at least, through depression of cellular IP 3 concentration although the mechanisms may not be identical. 6. 6. Quercetin and genistein, which attack different targets and different phases of the cell cycle, proved to be synergistic in OVCAR-5 cells. The impact of tiazofurin, genistein and quercetin is of interest because the drugs crucially inhibit the display of the neoplastic program of cells and lead to induced differentiation and apoptosis.

CORRELATED BIOCHEMICAL AND ULTRASTRUCTURAL CHANGES IN NITROGEN‐STARVED EUGLENA GRACILIS1

ABSTRACTGrowth of Euglena gracilis Z Pringsheim under photoheterotrophic conditions in a nitrogen‐deprived medium resulted in progressive loss of chloroplastic material until total bleaching of the cells occurred. Biochemical analysis and ultrastructural observation of the first stages of the starvation process demonstrated an early lag phase (from 0 to 9 h) in which cells increased in size, followed by a period of cell division, apparently supported by the mobilization of some chloroplastic proteins such as the photosynthetic CO2‐fixing enzyme ribulose‐1,5‐bisphosphate carboxylase/oxygenase. The degradation of the enzyme started after 9 h of starvation and was preceded by a transient concentration of this protein in pyrenoidal structures. Protein nitrogen and photosynthetic pigments as well as number of chloroplasts per cell decreased during proliferation through mere distribution among daughter cells. However, after 24 h, when cell division had almost ceased, there was a slow but steady decline of photosynthetic pigments. This was paralleled by observable ultrastructural changes including progressive loss of chloroplast structure and accumulation of paramylon granules and lipid globules in the cytoplasm. These findings reinforce the role of chloroplastic materials as a nitrogen source during starvation of E. gracilis in a carbon‐rich medium. The excess of ribulose‐1,5‐bisphosphate carboxylase/oxygenase acts as a first reservoir that, once exhausted, is superseded by the generalized disassembly of the photosynthetic structures, if the adverse environment persists more than 24 h.

Non-dystrophic 129 REJ mice are susceptible to i.p. infection with Listeria monocytogenes despite an ability to recruit inflammatory neutrophils to the peritoneal cavity

In this study we compared the host response toListeria monocytogenesin 129 REJ mice with listeria-resistant (C57Bl/6j) and susceptible (Balb/c) mouse strains. In all experiments mice were inoculated by the i.p. route. 129 REJ mice and Balb/c mice were sensitive to listeriosis whilst C57Bl/6j mice were relatively resistant to i.p. infection. Relatively large numbers of viable bacteria could be detected in the spleens of 129 REJ mice as early as 6 h following i.p. inoculation suggesting that dissemination of listeria from the peritoneal cavity is rapid in this mouse strain. This contrasted with Balb/c mice which exhibited an early lag phase during which only low numbers of bacteria could be isolated from the spleens of infected animals. In response to both proteose peptone and live listeria, 129 REJ mice demonstrated a greater capacity to recruit neutrophils to the peritoneal cavity than Balb/c and C57Bl/6j mice. In addition, inflammatory phagocytes from 129 REJ mice were as bactericidalin vitroas phagocytes from the Balb/c and C57Bl/6j strains. However,in vivo, inflammatory neutrophils elicited by proteose peptone prior to i.p. infection withL. monocytogeneswere not protective in the three mouse strains tested. Despite the apparent inadequacy of peritoneal neutrophils in controlling early bacterial proliferation, depletion of neutrophils in 129 REJ mice severely exacerbated i.p. infection withL. monocytogenes. The results indicate that neutrophils provide an inefficient but essential means of controlling early outgrowth of listeria in the peritoneal cavity of 129 REJ mice. The excessive inflammatory response seen in 129 REJ mice may facilitate the early dissemination ofL. monocytogenesfrom the peritoneal cavity to peripheral sites.

Experimental and simulated production of inulin by chicory and Jerusalem artichoke

Chicory and Jerusalem artichoke are candidate crops for inulin production. Their yield level and yearly stability of inulin yields are important criteria when deciding between them. A concise crop simulation model was used to estimate the yield potential of the two species and to quantify the effects on yields of some variations in characteristics found in the available cultivars and of some hypothetical improvements in the critical crop characteristics. In chicory the model attains at similar root yields as found in experiments. In Jerusalem artichoke the model properly describes the seasonal pattern of tuber growth, but the simulated yields show larger deviations from experimental results. Simulations of the root and tuber yields under the weather conditions of a rage of years indicate that the attainable yields in chicory are much higher than in Jerusalem artichoke. According to the simulations Jerusalem artichoke tuber yields are strongly affected by the temperature-related senescence of the leaves and by the ratio between structural stem dry matter and inulin stored temporarily in the stem. Chicory root yields are strongly related to early leaf expansion and to the shoot-root ratio. It is suggested that chicory breeders should concentrate on improving the assimilate supply to the first leaves in order to reduce the early lag phase in leaf area growth, rather than increasing the growth rate during the temperature related exponential phase of leaf expansion.

Multiplication of Xanthomonas campestris pvs secalis and translucens in host and non‐host plants (rye and barley) and development of water soaking1

Multiplication of the rye‐specific and barley‐specific pathovars secalis and translucens, respectively, of Xanthomonas campestris (earlier named Xanthomonas translucens f. sp. cerealis) was determined in two rye and two barley cultivars. Fresh bacterial cultures were suspended in boiled tap water, adjusted to about 3 × 107 cfu ml−1 and infiltrated into the mesophyll of young leaves. In the compatible combinations, a generation time of 1.7–2.1 h was determined. Significantly higher multiplication rates than in incompatible combinations were not observed before 3 or 4 days after inoculation. At this time, the first small water‐soaked areas appeared. Bacteria multiplied up to the seventh day after inoculation, reaching a maximum of at least 109 cells per cm2, the same time at which the water‐soaked areas had reached their maximum size. Thus, the rate of bacterial multiplication paralleled with the development of the water‐soaked areas. In the incompatible combinations, nearly the same generation times were detected during the exponential growth phase. However, water‐soaked areas did not appear, and the final bacterial concentration ranged from 1:1000 to 1:100 of that determined in compatible combinations. A distinct early lag phase, characterized by a decrease of the bacterial population down to 1:100 of the starting concentration, was detected in all cases.