Production of cytochalasins C,D & E from dematiaceous hyphomycetes

One hundred isolates of 27 species belonging to 13 genera of dematiaceous hyphomycetes were screened for production of cytochalasins C, D and E. Only two isolates ofBipolaris neergaardii and one isolate ofPhoma herbarum produced cytochalasins C and D. Also, cytochalasin E was produced by two isolates ofAlternaria chlamydospora, and one isolate ofCochliobolus tuberculatus. This is the first report about the production of cytochalasins C, D and E by these species of dematiaceous hyphomycetes.     

Determination of Kainic Acid-Induced Release of Nitric Oxide Using a Novel Hemoglobin Trapping Technique with Microdialysis

We have demonstrated the usefulness of a novel hemoglobin-trapping technique to quantify nitric oxide (NO) concentrations in vivo. Concentric microdialysis probes were implanted into the hippocampus of rats under urethane anesthesia and perfused with 1 μM oxyhemoglobin in artificial CSF to sequester NO in extracellular fluid. The concentration of methemoglobin was then determined spectrophotometrically. The basal level of NO in hippocampus was 2.2 ± 0.5 nM(in vitro sensitivity of the probe was 0.2 nM). Administration of 13 mg/kg, i.p., of kainic acid (KA) produced a maximal 5.3-fold increase at 100 min in NO levels (11.8 ± 0.2 nM). This response was significantly attenuated by pretreatment with the NO synthase inhibitor N-monomethyl-L-arginine (50 mg/kg, 30 min before KA). These results demonstrate that a microdialysis probe using a novel hemoglobin-trapping technique possesses adequate sensitivity to determine the basal levels of NO and document the ability of KA to increase these levels via a NO synthase-mediated mechanism.     

Random forest and live single-cell metabolomics reveal metabolic profiles of human macrophages upon polarization

Human macrophages are innate immune cells with diverse, functionally distinct phenotypes, namely, pro-inflammatory M1 and anti-inflammatory M2 macrophages. Both are involved in multiple physiological and pathological processes, including would healing, infection, and cancer. However, the metabolic differences between these phenotypes are largely unexplored at single-cell resolution. To address this knowledge gap, an untargeted live single-cell mass spectrometry-based metabolomic profiling coupled with a machine-learning data analysis approach was developed to investigate the metabolic profile of each phenotype at the single-cell level. Results show that M1 and M2 macrophages have distinct metabolic profiles, with differential levels of fatty acyls, glycerophospholipids, and sterol lipids, which are important components of plasma membrane and involved in multiple biological processes. Furthermore, we could discern several putatively annotated molecules that contribute to inflammatory response of macrophages. The combination of random forest and live single-cell metabolomics provided an in-depth profile of the metabolome of primary human M1 and M2 macrophages at the single-cell level for the first time, which will pave the way for future studies targeting the differentiation of other immune cells.     

Impact of peroxisome proliferator activated receptor agonist drugs in a model of nephrotoxicity in rats

Doxorubicin (DOX) is one of the basic anticancer drugs, nonetheless its use is restricted due to noxious side effects. Kidney failure is one of the main side effects that restrict its medical use. The current study assessed the nephroprotective effects of fenofibrate and pioglitazone against the renal injury induced by doxorubicin in rats and illustrated the probable mechanisms underlying these protective effects. For this purpose, Male Sprague–Dawley rats weighing (200–230 g) were allocated into seven groups treated for 15 days as following: control (50% corn oil + 50% DMSO p.o), fenofibrate (100 mg/kg p.o) and pioglitazone (10 mg/kg p.o) as well as four groups of DOX (15 mg/kg i.p on 11th day). DOX groups included DOX alone and DOX with protective drugs fenofibrate, pioglitazone or both of them. As a result of doxorubicin nephrotoxicity; serum creatinine and blood urea nitrogen were remarkably elevated. Moreover, renal glutathione was significantly reduced while tissue lipid peroxidation malondialdehyde, tumor necrosis factor-α, nuclear factor-kappa B p65 (NF-κB p65), interleukin-1β, p38 mitogen activated protein kinase (p38-MAPK) and caspase-3 (Casp-3) were significantly augmented. Treatment with fenofibrate and pioglitazone either alone or in combination markedly attenuated DOX-induced injury by suppression of oxidative stress, inflammation and apoptosis. The above-mentioned biochemical markers were affirmed by histological assessment. In conclusion, fenofibrate, pioglitazone, and their combination possess potential prophylactic effects against doxorubicin-induced renal injury through modulation of p38-MAPK/NF-κB p65 pathway with superiority to the combination.     

Cytotoxicity and mutagenicity of aflatoxin dichloride in normal and repair deficient diploid human fibroblasts

The cytotoxic and mutagenic effect of aflatoxin B1-dichloride (AFB1-Cl2), a direct-acting carcinogen which is a model for the proposed ultimate reactive metabolite of AFB1 (the 2,3-epoxide), was compared in normal, repair-proficient, diploid human fibroblasts and in complementation Group A xeroderma pigmentosum cells (XP12BE) which are virtually incapable of excision repair of DNA damage induced by ultraviolet radiation, the 7,8-diol-9,10-epoxide of benzo[alpha]pyrene, and several reactive aromatic amide derivatives. The XP cells were significantly more sensitive than normal to the cytotoxic and mutagenic effects of AFB1-Cl2, not only as a function of concentration administered but also of the number of AFB1-Cl2 residues initially bound to DNA. Cytotoxicity was determined from survival of colony-forming ability; resistance to 6-thioguanine was the genetic marker used for mutagenicity. We compared the rate of loss of AFB1-Cl2-DNA adducts from cells treated and held in the non-dividing state (confluent) over several days, as well as their ability to recover from the potentially mutagenic and/or cytotoxic effects of the agent. AFB1-Cl2 residues were lost from both strains of cells and both exhibited a gradual increase in survival. However, the rate of loss of adducts from the DNA in the normal cells was more rapid than in XP cells and they exhibited recovery from higher doses of AFB1-Cl2 than XP cells. The major primary DNA adduct formed in the human cells and in isolated DNA was a chemically unstable guanine derivative which could undergo a change in structure with time posttreatment to form a more stable secondary adduct. The cytotoxic effect of AFB1-Cl2 was highly correlated with the presence of either of these guanine adducts. Evidence suggests that the primary adduct is an N7-guanine adduct. The kinetics of the loss of this guanine and its transformation into the more stable secondary adduct resembled that reported recently for the major primary DNA adduct formed by the reaction of AFB1 at the N-7 position of guanine in the DNA of normal and XP cells and its transformation into the putative AFB1-ring opened triamino pyrimidyl structure.     

Extent of excision repair before DNA synthesis determines the mutagenic but not the lethal effect of UV radiation

Excision repair-proficient diploid fibroblasts from normal persons (NF) and repair-deficient cells from a xeroderma pigmentosum patient (XP12BE, group A) were grown to confluence and allowed to enter the G₀ state. Autoradiography studies of cells released from G₀ after 72 h and replated at lower densities (3?9 × 10³ cells/cm²) in fresh medium containing 15% fetal bovine serum showed that semiconservative DNA synthesis (S phase) began ～24 h after the replating. To determine whether the time available for DNA excision repair between ultraviolet irradiation (254 nm) and the onset of DNA synthesis was critical in determining the cytotoxic and/or mutagenic effect of UV in human fibroblasts, we released cultures of NF or XP12BE cells from G₀, allowed them to reattach at lower densities, irradiated them in early G₁ (～18 h prior to the onset of S) or just prior to S phase, and assayed the frequency of mutations to 6-thioguanine resistance and the survival of colony-forming ability. The XP12BE cells, which are virtually incapable of excising UV-induced DNA lesions, showed approximately the same frequency of mutations and survival regardless of the time of UV irradiation. In NF cells, the slope of the dose response for mutations induced in cells irradiated just prior to S was about 7-fold steeper than that of cells irradiated 18 h earlier. However, the two sets of NF cells showed no significant difference in survival. Neither were there significant differences in the survival of NF cells released from G₀, plated at cloning densities and irradiated as soon as they had attached and flattened out (～20 h prior to S) or 4, 8, 12, 16, 20 or 24 h later. We conclude that the frequency of mutations induced by UV is dependent upon the number of unexcised lesions remaining at the time of semi-conservative DNA replication. However, the amount of time available for excision of potentially cytotoxic lesions is not determined primarily by the period between irradiation and the onset of S phase.     

The basic fibroblast growth factor-saporin mitotoxin acts through the basic fibroblast growth factor receptor.

We have confirmed the hypothesis that a mitotoxin resulting from the conjugation of basic fibroblast growth factor and saporin exerts its cytotoxic effect through specific interaction with the basic fibroblast growth factor (FGF) receptor. Accordingly, the mitotoxin stimulates tyrosine phosphorylation of the 90 kD substrate that characterizes the initial cellular response to basic FGF. Cross-linking experiments show that radio-labeled basic fibroblast growth factor-saporin (FGF-SAP) binds to the receptor. Suramin, an inhibitor of growth factor receptor binding, inhibits the cytotoxicity of basic FGF-SAP. In a study of 4 different cell types, there is a decrease in the ED50 of the mitotoxin as the receptor number per cell increases. We have verified the cytotoxicity of the mitotoxin in 3 different assay systems. As expected, it is effective in the inhibition of protein synthesis and DNA synthesis, as well as of cell count. Binding of basic FGF-SAP which will result in cytotoxicity occurs very rapidly; 5 minutes of incubation of 10 nM basic FGF-SAP with cells results in 80% inhibition of cell count. The in vitro data indicate that the basic FGF-SAP is a receptor specific and potent suicide antagonist of basic FGF. Its potential as an anti-FGF for therapeutic and research uses in vivo is discussed.     

Serial natriuretic response to atrial peptide in preascitic bile duct ligated dogs

We determined if nine precirrhotic unanaesthetized dogs with chronic bile duct ligation (CBDL) responded uniformly to atrial natriuretic peptide (ANF) by infusing this peptide sequentially over 8-12 weeks at 175 ng.kg-1.min-1 and observing the natriuretic response. ANF was administered every 2 weeks post-CBDL until the 8th week and given again during the cirrhotic phase with ascites present (10-12 weeks post-CBDL). Sodium balance studies were conducted at similar time intervals. During the control period and at weeks, 2, 6, and 8 post-CBDL all dogs responded to ANF with a significant change in sodium excretion (delta UNaV, 50-240 mu equiv./min). At these times, all dogs were in sodium balance. At week 4 and during the ascitic period, heterogeneity of response to ANF was observed. In the former interval, five dogs responded (delta UNaV,75-230 mu equiv./min) and four did not, while in the latter interval, five dogs responded (delta UNaV, 50-240 mu equiv./min) and three did not (one dog died). In both time periods, there was severe urinary sodium retention (daily UNaV, 11 +/- 3 and 2 +/- 1 mequiv./day, respectively) while the dogs were ingesting 45 mequiv.Na+/day. The heterogeneity of natriuretic response was not correlated to plasma immunoreactive ANF, renin, or aldosterone levels. Plasma volume was significantly expanded from control during both intervals. We conclude that there is transient sodium retention during the 4th week post-CBDL, and that this period is associated with the heterogeneity of natriuretic response to ANF, despite the absence of ascites or edema.