Growth-inhibiting Factor Research Articles

Disruption of phosphate metabolism and sterol transport-related genes conferring yeast resistance to vanillin and rapid ethanol production

Vanillin is a potent growth-inhibiting factor in Saccharomyces cerevisiae during lignocellulose biorefineries. Here, a haploid gene-deletion library was screened to search for vanillin-tolerant mutants and explain the possible tolerance mechanisms. Twenty-two deletion mutants were identified. The deleted genes in these mutants were involved in phosphate and inositol polyphosphate metabolism and intracellular sterol transport. Activation of the phosphate signaling pathway is not conducive to yeast against the pressure of vanillin. Furthermore, the findings indicate the role of inositol polyphosphates in altering vanillin tolerance by regulating phosphate metabolism. Meanwhile, reducing the transport of sterols from the plasma membrane enhanced tolerance to vanillin. In the presence of vanillin, the representative yeast deletions, pho84Δ and lam3Δ, showed good growth performance and promoted rapid ethanol production. Overall, this study identifies robust yeast strain alternatives for ethanol fermentation of cellulose and provides guidance for further genomic reconstruction of yeast strains.

Estimation of the growth inhibiting factor based on the correlation between soil humus content and growth of sugar beet estimated from satellite images to identify poor drainage fields

Estimation of the growth inhibiting factor based on the correlation between soil humus content and growth of sugar beet estimated from satellite images to identify poor drainage fields

Diclofenac and atrazine restrict the growth of a synchronous Chlamydomonas reinhardtii population via various mechanisms

Non-steroidal anti-inflammatory drug diclofenac (DCF) is commonly found in freshwater bodies and can have adverse effects on non-target organisms. Among the studies on DCF toxicity, several ones have reported its harmful effects on plants and algae. To gain a better understanding of the mechanisms of DCF toxicity towards green algae, we used a synchronous Chlamydomonas reinhardtii cc-1690 culture and compared DCF (135 mg/L) effects with effects caused by atrazine (ATR; 77.6 μg/L), an herbicide with a well-known mechanism of toxic action. To achieve our goal, cell number and size, photosynthetic oxygen consumption/evolution, chlorophyll a fluorescence in vivo, H2O2 production by the cells, antioxidative enzymes encoding genes expression were analyzed during light phase of the cell cycle.We have found, that DCF and ATR affect C. reinhardtii through different mechanisms. ATR inhibited the photosynthetic electron transport chain and induced oxidative stress in chloroplast. Such chloroplastic energetics disruption indirectly influenced respiration, the intensification of which could partially mitigate low efficiency of photosynthetic energy production. As a result, ATR inhibited the growth of single cell leading to limitation in C. reinhardtii population development. In contrast to ATR-treated algae, in DCF-treated cells the fraction of active PSII reaction centers was diminished without drastic changes in electron transport or oxidative stress symptoms in chloroplast. However, significant increase in transcript level of gene encoding for mitochondria-located catalase indicates respiratory processes as a source of H2O2 overproduced in the DCF-treated cells. Because the single cell growth was not strongly affected by DCF, its adverse effect on progeny cell number seemed to be related rather to arresting of cell divisions. Concluding, although the DCF phytotoxic action appeared to be different from the action of the typical herbicide ATR, it can act as algal growth-inhibiting factor in the environment.

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Phosphate-Catalyzed Hydrogen Peroxide Formation from Agar, Gellan, and κ-Carrageenan and Recovery of Microbial Cultivability via Catalase and Pyruvate.

Previously, we reported that when agar is autoclaved with phosphate buffer, hydrogen peroxide (H2O2) is formed in the resulting medium (PT medium), and the colony count on the medium inoculated with environmental samples becomes much lower than that on a medium in which agar and phosphate are autoclaved separately (PS medium) (T. Tanaka et al., Appl Environ Microbiol 80:7659-7666, 2014, https://doi.org/10.1128/AEM.02741-14). However, the physicochemical mechanisms underlying this observation remain largely unknown. Here, we determined the factors affecting H2O2 formation in agar. The H2O2 formation was pH dependent: H2O2 was formed at high concentrations in an alkaline or neutral phosphate buffer but not in an acidic buffer. Ammonium ions enhanced H2O2 formation, implying the involvement of the Maillard reaction catalyzed by phosphate. We found that other gelling agents (e.g., gellan and κ-carrageenan) also produced H2O2 after being autoclaved with phosphate. We then examined the cultivability of microorganisms from a fresh-water sample to test whether catalase and pyruvate, known as H2O2 scavengers, are effective in yielding high colony counts. The colony count on PT medium was only 5.7% of that on PS medium. Catalase treatment effectively restored the colony count of PT medium (to 106% of that on PS medium). In contrast, pyruvate was not as effective as catalase: the colony count on sodium pyruvate-supplemented PT medium was 58% of that on PS medium. Given that both catalase and pyruvate can remove H2O2 from PT medium, these observations indicate that although H2O2 is the main cause of reduced colony count on PT medium, other unknown growth-inhibiting substances that cannot be removed by pyruvate (but can be by catalase) may also be involved.IMPORTANCE The majority of bacteria in natural environments are recalcitrant to laboratory culture techniques. Previously, we demonstrated that one reason for this is the formation of high H2O2 levels in media prepared by autoclaving agar and phosphate buffer together (PT medium). In this study, we investigated the factors affecting H2O2 formation from agar. H2O2 formation is pH dependent, and ammonium ions promote this phosphate-catalyzed H2O2 formation. Amendment of catalase or pyruvate, a well-known H2O2-scavenging agent, effectively eliminated H2O2 Yet results suggest that growth-inhibiting factor(s) that cannot be eliminated by pyruvate (but can be by catalase) are present in PT medium.

Impact of climate change on filarial vector, Culex quinquefasciatus and control using bacterial pesticide, spinosad

ObjectiveTo show the effect of temperature on the biology of Culex quinquefasciatus and also to show the effect of the bacterial pesticide, spinosad on developmental stages of the filarial vector. MethodsA laboratory colony of mosquito larvae was used for the larvicidal activity of temperature and spinosad. Twenty-five numbers of first, second, third, fourth instar larvae were introduced into the 500 mL glass beaker containing 250 mL of de-chlorinated water with desired temperatures (16°C, 20°C, 24°C, 28 °C, 32 °C, 36 °C), similarly spinosad, at different concentrations. The development was observed for every 24 h. ResultsThe results showed that the rise in temperature acts as a growth inhibiting factor for mosquitoes. And no development was found in the temperature below 16 °C and above 36 °C. The hatchability was increased as the temperature was increased up to 32 °C, after which eclosion rates dropped gradually. Conclusions32 °C was obtained as the maximum sustainable temperature and after which the developmental rate was gradually reduced. The optimal temperature for development was lower than the temperatures at which development was quickest. The bacterial pesticide spinosad showed that it is an effective mosquito control agent and can be used for further integrated pest management programmes.

Intracellular tension in periosteum/perichondrium cells regulates long bone growth

Perichondrium/periosteum is involved in regulating long bone growth. Long bones grow faster after removal or circumferential division of periosteum. This can be countered by culturing them in conditioned medium from perichondrium/periosteum cells. Because both complete removal and circumferential division are effective, we hypothesized that perichondrium/periosteum cells require an intact environment to release the appropriate soluble factors. More specifically, we propose that this release depends on their ability to generate intracellular tension. This hypothesis was explored by modulating the ability of perichondrium/periosteum cells to generate intracellular tension and monitoring the effect thereof on long bone growth. Perichondrium/periosteum cells were cultured on substrates with different stiffness. The medium produced by these cultures was added to embryonic chick tibiotarsi from which perichondrium/periosteum was either stripped or left intact. After 3 culture days, long bone growth was proportionally related to the stiffness of the substrate on which perichondrium/periosteum cells were grown while they produced conditioned medium. A second set of experiments demonstrated that the effect occurred through expression of a growth-inhibiting factor, rather than through the reduction of a stimulatory factor. Finally, evidence for the importance of intracellular tension was obtained by showing that the inhibitory effect was abolished when perichondrium/periosteum cells were treated with cytochalasin D, which disrupts the actin microfilaments. Thus, we concluded that modulation of long bone growth occurs through release of soluble inhibitors by perichondrium/periosteum cells, and that the ability of cells to develop intracellular tension through their actin microfilaments is at the base of this mechano-regulated control pathway.

Antibiotic effects of  three strains of chrysophytes (Ochromonas, Poterioochromonas) on freshwater bacterial isolates

We investigated the antibiotic effects of extracts of freeze-dried biomass and culture supernatants from the mixotrophic chrysophyte species Ochromonas danica, Poterioochromonas sp. strain DS, and Poterioochromonas malhamensis on bacterial strains isolated from lake water. Methanolic biomass extracts inhibited the growth of all tested strains, albeit to a different extent, whereas aqueous biomass extracts only affected bacteria of the genus Flectobacillus. The antibiotic action of supernatants from flagellate cultures could be mostly attributed to lipophilic substances, but the growth of bacteria affiliated with Flectobacillus and Sphingobium was also affected by hydrophilic compounds. A comparison of biomass extracts from light- and dark-adapted cultures of Poterioochromonas sp. strain DS showed that the growth-inhibiting factor was unrelated to chlorophyll derivatives. Supernatants from a dark-adapted, phagotrophically grown flagellate culture had stronger antibiotic effects and affected more bacterial strains than the supernatant from a light-adapted culture. Significant growth reduction of a Flectobacillus isolate was already induced by extremely low concentrations of lipophilic extracts from these supernatants. Our results show that metabolites of the studied flagellates - either released actively or during cell lysis - may selectively affect the growth of some aquatic bacteria even in very small doses and thus potentially affect microbial community composition. Moreover, the antibiotic potential of mixotrophic chrysophytes may change with their nutritional mode.

Generating Disulfides in Multicellular Organisms: Emerging Roles for a New Flavoprotein Family

Generating Disulfides in Multicellular Organisms: Emerging Roles for a New Flavoprotein Family

Bone marrow stroma damage induced by chemotherapy for acute lymphoblastic leukemia in children.

Several studies have suggested a role of bone marrow stroma injury in long-term chemotherapy-induced hematopoietic failure. To evaluate whether bone marrow microenvironment is altered by chemotherapy for acute lymphoblastic leukemia (ALL) and to determine its contribution to postchemotherapy anemia, we investigated the ability of stroma from children receiving maintenance chemotherapy for ALL to support hematopoiesis. Long-term bone marrow cultures (LTBMC) were established with bone marrow cells either from ALL children under therapy (n = 24) or from control subjects (n = 19). Nonadherent cells and colony forming units-granulocytic monocytic (CFU-GM) output in LTBMC did not differ between patients and controls. In contrast, burst forming unit-erythroid (BFU-E) numbers were lower in patient LTBMC (p = 0.013). Co-cultures of normal CD34+ cells and preformed patient or control stromas showed significantly reduced hematopoietic supportive capabilities of patient stromas: both CFU-GM and BFU-E were reduced (p = 0.002 and 0.046, respectively). In addition, supernatants (SN) of patients' LTBMC inhibited normal BFU-E growth compared with SN of normal LTBMC. Transforming growth factor (TGF)-beta1 levels were increased in patient cultures (p = 0.0039) and inversely correlated with BFU-E produced in LTBMC (r = -0.36, p = 0.04). Neutralization of TGF-beta1 significantly increased the BFU-E output of patient LTBMC (p = 0.0078). In contrast, macrophage inflammatory peptide (MIP)-1alpha levels were lower in SN of patients compared with controls (p = 0.015). Thus, chemotherapy for ALL induces functional deregulation within bone marrow stromal cells with an increase in the growth-inhibiting factor TGF-beta1, together with a decrease in MIP-1alpha, which might contribute to hematopoietic toxicity.

Effects of the auxin-inhibiting substances raphanusanin and benzoxazolinone on apical dominance of pea seedlings

The effects of the auxin-inhibiting substances raphanusanin ((3R*,6S*)-3-[methoxy (methylthio) methyl]-2-pyrrolidinethione, raphanusanin B)and benzoxazolinone (6-methoxy-2-bezoxazolinone, MBOA) on apical dominance of pea(Pisum sativum L. cv. Alaska) seedlings were studied.Application of raphanusanin B or MBOA to the apical bud, internode, or lateralbud of pea seedlings released apical dominance in either intact orindole-3-acetic acid (IAA )-treated, decapitated plants. These results suggestthat the auxin-inhibiting substances raphanusanin B and MBOA have activity inreleasing apical dominance. Conversely, the auxin transport inhibitors2,3,4-triiodobenzoic acid (TIBA) and 1-naphthylphthalamic acid (NPA) did notstimulate lateral bud growth when they were applied directly to the lateralbud,although application to the apical bud or internode released apical dominance.Therefore, the mode of action of raphanusanin B and MBOA in apical dominance isclearly different from that of auxin transport inhibitors. Raphanusanin B andMBOA may suppress the synthesis of growth-inhibiting factor(s) of the lateralbud induced by endogenous auxin transported from the apical bud or exogenouslyapplied auxin, and/or the action of the factor(s).

Evaluation of raw and heated velvet beans (Mucuna pruriens) as feed ingredients for broilers

Velvet bean plants (Mucuna pruriens) are used widely outside the U.S. as a cover crop. The beans (VB), high in protein, contain toxic substances that possibly can be destroyed by heating. Few data are available on the use of VB in poultry nutrition. We examined the effects of raw and dry-roasted VB on broiler performance in two experiments. In Experiment 1, 10, 20, and 30% raw VB were substituted into nutritionally balanced rations fed 0 to 42 d of age. Raw VB caused progressive reductions in growth; at 42 d of age, broilers fed 30% VB weighed 39% of controls. Feed intake declined significantly only with 30% VB. Feed efficiency decreased significantly with 20 and 30% VB. In Experiment 2, 10% raw VB and 10, 20, and 30% heated VB were fed 0 to 42 d. With 10% raw VB, broilers grew significantly slower but feed intake was unchanged. Inclusion of 10% heated VB allowed better growth than raw VB, and by 42 d of age, growth was not significantly different from that of controls. At 20 and 30%, heated VB promoted much better growth and efficiency than raw VB in Experiment 1, but values were significantly lower than those of controls. With 30% heated VB, broilers grew to 66% of control, a marked improvement over raw VB. Carcass yield was unaffected. Trypsin inhibitor activity but not L-3,4-dihydroxyphenylalanine (L-DOPA) in VB was destroyed by heating. We conclude that dry heating of VB partially destroys its growth-inhibiting factor(s), allowing successful use of 10% heated VB in broiler rations. Higher levels of heated VB reduced broiler performance, although results were much better than those of raw VB.

Antitumor Effects of IL-6 on Murine Liver Tumor Cells in vivo

IL-6 is a pleiotropic cytokine that is capable of modulating the diverse functions of hepatocytes such as acute phase responses and inflammation in the liver. To learn its antitumor effects in vivo, the cDNA of IL-6 was transfected into murine liver cells, TIB cells. IL-6-transfected TIB cells (TIB73-IL-6 or TIB75-IL-6) produced much higher levels of IL-6 compared with vector-transfected TIB cells (TIB73-vec or TIB75-vec). To investigate the effects of IL-6 on TIB tumor growth in vivo, IL-6-transfected TIB cells or vector-transfected TIB cells were injected subcutaneously into syngeneic mice. Vector-transfected TIB cells grew rapidly 3 weeks after injection, but IL-6-transfected TIB cells did not grow at all for up to 6 weeks. Pathologically, IL-6-transfected TIB cells demonstrated a severe necrosis and apoptotic pattern. Taken together, these results indicate that IL-6 functions as a growth inhibiting factor in vivo, and another biological role of IL-6 in the liver is suggested.

Antitumor effects of IL-6 on murine liver tumor cells in vivo.

IL-6 is a pleiotropic cytokine that is capable of modulating the diverse functions of hepatocytes such as acute phase responses and inflammation in the liver. To learn its antitumor effects in vivo, the cDNA of IL-6 was transfected into murine liver cells, TIB cells. IL-6-transfected TIB cells (TIB73-IL-6 or TIB75-IL-6) produced much higher levels of IL-6 compared with vector-transfected TIB cells (TIB73-vec or TIB75-vec). To investigate the effects of IL-6 on TIB tumor growth in vivo, IL-6-transfected TIB cells or vector-transfected TIB cells were injected subcutaneously into syngeneic mice. Vector-transfected TIB cells grew rapidly 3 weeks after injection, but IL-6-transfected TIB cells did not grow at all for up to 6 weeks. Pathologically, IL-6-transfected TIB cells demonstrated a severe necrosis and apoptotic pattern. Taken together, these results indicate that IL-6 functions as a growth inhibiting factor in vivo, and another biological role of IL-6 in the liver is suggested.

Studies on maintenance of vector pBK2 in Corynebacterium acetoacidophilum by co-culture experiments in a continuous bioreactor

A plasmid vector pBK2 was tested for maintenance in Corynebacterium acetoacidophilum and found to be 100% stable for 90 generations. In co-culture experiments in absence of any selection pressure it was able to cause washout of plasmid free cells. Deletion mutagenesis of the plasmid was done to identify the regions of DNA responsible for its stable maintenance. This led to the identification of a region of DNA in pBB1, an endogenous plasmid used in the construction of pBK2, which is responsible for providing a growth rate advantage to the plasmid containing cell in co-culture with plasmid-free cells. To explain this observed stability we postulate the production of a “growth-inhibiting factor” by the plasmid-containing cells. Inclusion of a death rate term for the plasmid free cells in the computer simulation of the growth kinetics in a chemostat successfully predicts, depending upon the magnitude of the kinetic parameters, a stable coexistence of the two or washout of the plasmid-free cells, an observation that matches the experimental data. Because of its stable maintenance and its ability to select against plasmid-free cells this host-vector system is a natural candidate for cloning and use in large-scale fermentations.

Activation of Cell Growth Inhibitor by Ectoprotein Kinase-mediated Phosphorylation in Transformed Mouse Fibroblasts

Our previous studies have shown that exogenous ATP induces cell growth inhibition in transformed mouse fibroblasts, 3T6 cells, whereas the growth of their nontransformed counterparts, Swiss 3T3 cells, is only slightly affected. In this study a similar selective, ATP-induced growth inhibition was found in Balb/c SV40-3T3 cells and in primary cultures of adenovirus-transformed murine fibroblasts. The inhibitory activity was found in the conditioned media of ATP-treated cultures. Several lines of evidence have shown that ectoprotein kinase (ecto-PK) plays a major role in the ATP-induced growth inhibition. (a) There is a good correlation between the activity of ecto-PK and the ability of ATP to induce cell growth inhibition. (b) The removal of the ecto-PK from the cell surface prevents the ATP-induced growth inhibition. (c) Addition of the removed enzyme to the cell culture reconstitutes the ability of ATP to induced growth inhibition. (d) Serum-containing, or serum-free, conditioned media from untreated cultures gain an inhibitory activity after their phosphorylation, and dephosphorylation of conditioned media from ATP-treated cultures results in the loss of the inhibitory activity. (e) Growth medium by itself does not inhibit cell proliferation after its phosphorylation. The findings described in d and e indicate, as well, that the ATP-induced growth inhibitor is produced by the cells. The putative inhibitor was found to be a protein, with an apparent molecular mass of 13 kDa. The selectivity of the inhibition for transformed cells is due to the higher level of ecto-PK in these cells, as well as to their higher susceptibility to the inhibitor, as compared with their non-transformed counterparts.

Presence of immunoassayable transforming growth factor‐β1 (tgf‐β1) in breast cyst fluid (BCF): Relationship with the intracystic electrolyte and epidermal‐growth‐factor (EGF) content

We evaluated the presence and distribution of transforming growth factor-beta 1 (TGF-beta 1) in breast cyst fluid (BCF), and its relationship with intracystic epidermal growth factor (EGF). EGF and TGF-beta 1 were determined by radioimmunoassay on 47 BCFs (27 of the Na+/K+ < 3 type and 20 of the Na+/K+ > 3 type). As expected, EGF levels were inversely correlated with the Na+/K+ ratio, and were consequently higher in Na+/K+ < 3 cysts as compared with Na+/K+ > 3 cysts, (p < 0.005). By contrast, TGF-beta 1 levels were directly correlated with the Na+/K+ ratio (p < 0.01), being higher in Na+/K+ > 3 cysts, though not significantly (p = 0.057). A significant negative relationship was found between EGF and TGF-beta 1 concentration. When the analysis was performed separately in the 2 cyst sub-populations, EGF and TGF-beta 1 were found to be negatively and significantly correlated in Na+/K+ < 3 cysts only (p < 0.01). Our results demonstrate that Na+/K+ < 3 cysts contain high levels of EGF, a growth-stimulating factor, and very low levels of TGF-beta 1, a growth-inhibiting factor. This may provide an explanation for the higher risk of breast cancer observed in women with Na+/K+ < 3 cysts. Our results also suggest that EGF accumulation in this type of cysts might be regulated by TGF-beta 1.

Growth modulation of human tumor cells by a growth-inhibiting activity derived from tumorigenic V79 Chinese hamster cells.

A growth-inhibiting activity was identified in supernatants of the neoplastic V79 Chinese hamster cell line based on its ability to inhibit the proliferation of the same cell line. The partially purified activity, provisionally termed "growth inhibiting factor" (GIF) activity, inhibited the growth of a wide variety of human tumor cells, but not various normal human fibroblasts. This species-nonspecific activity was reversible, saturable, and highly potent in tumorigenic cell lines, and was noted in both monolayer culture and in soft agar. The inhibitory activity of GIF was also exhibited in a chemically defined serum-free medium supplemented with insulin and transferrin. GIF activity was stable to acid, heat, trypsin, and dithiothreitol but sensitive to alpha-chymotrypsin. The pattern of growth modulation by GIF on V79 cells was apparently different from those exhibited by bifunctional peptides such as transforming growth factor-beta, tumor necrosis factor-alpha, and interleukin-1-alpha. In addition, GIF activity cannot be ascribed to these cytokines based on the physicochemical and immunologic properties. Although GIF has yet to be purified to homogeneity, these data suggest that GIF might be a novel growth regulator which has a critical role in regulating growth of V79 cells. The growth modulation of tumor cells by this tumor-derived growth inhibiting activity suggested the presence of an autocrine growth regulatory mechanism even in tumor cells.

Feeding value of raw or heated grain amaranth germplasm

Four growth experiments were conducted with weanling male rats to determine the nutritional value of grain from four cultivars of Amaranthus species ( Amaranthus cruentus ‘477913’ and Amaranthus hypochondriacus ‘1023’, ‘K343’ and ‘R158’). Feed was offered ad libitum to individually caged animals for 12–14 days. Amaranth was ground and given raw, dry-heated (2 h at 100°C), moist-heated (10 min in boiling water) or soaked (16 h in water) as the sole source of protein and energy. Rats fed on raw, dry-heated or soaked amaranth gained weight for the first 6 days, then lost weight; rats fed on moist-heated amaranth gained weight at a rate equal to that of rats fed on a maize and soya-bean meal diet containing 16% protein. When rats were fed on raw or moist-heated ‘R158’ or black seed separated from ‘K343’, body weight gain with moist-heated amaranth was equal to that produced by the maize-soya bean meal diet, confirming the high nutritive value of moist-heated amaranth (including the black seeds separated from ‘K343’). This occurred despite the higher cell wall content of black seeds of ‘K343’ (27%) than of the unseparated ‘K343’ cultivar (12%). The overall results show that cultivars and lines of A. hypochondriacus and A. cruentus have a growth-inhibiting factor (or factors) labile to moist heat.

Conditioned medium of human nasopharyngeal carcinoma epithelioid cell line cne1 contained the activities of transformed growth-inhibiting factors

The hormone defined serum free conditioned medium (SFCM) of human nasopharyngeal carcinoma epithelioid cell line (CNE 1) was assaied by both the [3H] thymidine incorporation test and the soft agar test. It was found that the SFCM can stimulate the growth of long-term serum free cultured CNE 1 cells in accordence with the fact that the growth rate of long-term serum free cultured CNE 1 cells was directly proportional to the plating density. Alternatively 5% SFCM can inhibit the growth of short-term serum free cultured CNE 1 cells by 51% in which the indicator cell may remained the responsive state of growing in the serum supplemented medium to the effector of interest. Furthermore, SFCM resulted in inhibition of anchorage-independent growth of CNE 1 cells and A431 cells. Also in soft agar test, SFCM can reduce the colony formation of NRK-49F cells in the presence of EGF or EGF plus TGF-beta. These finding suggested that CNE 1 can autocrine growth stimulating factor(s) and growth inhibiting factor(s) in the serum free medium, the latter can strongly reverse malignant phenotypies of CNE 1 and A431 cells in serum supplemented surrounding.