Perampanel, an antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, for the treatment of epilepsy: studies in human epileptic brain and nonepileptic brain and in rodent models.

Perampanel [Fycompa, 2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)benzonitrile hydrate 4:3; Eisai Inc., Woodcliff Lake, NJ] is an AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor antagonist used as an adjunctive treatment of partial-onset seizures. We asked whether perampanel has AMPA receptor antagonist activity in both the cerebral cortex and hippocampus associated with antiepileptic efficacy and also in the cerebellum associated with motor side effects in rodent and human brains. We also asked whether epileptic or nonepileptic human cortex is similarly responsive to AMPA receptor antagonism by perampanel. In rodent models, perampanel decreased epileptic-like activity in multiple seizure models. However, doses of perampanel that had anticonvulsant effects were within the same range as those engendering motor side effects. Perampanel inhibited native rat and human AMPA receptors from the hippocampus as well as the cerebellum that were reconstituted into Xenopus oocytes. In addition, with the same technique, we found that perampanel inhibited AMPA receptors from hippocampal tissue that had been removed from a patient who underwent surgical resection for refractory epilepsy. Perampanel inhibited AMPA receptor-mediated ion currents from all the tissues investigated with similar potency (IC50 values ranging from 2.6 to 7.0 µM). Cortical slices from the left temporal lobe derived from the same patient were studied in a 60-microelectrode array. Large field potentials were evoked on at least 45 channels of the array, and 10 µM perampanel decreased their amplitude and firing rate. Perampanel also produced a 33% reduction in the branching parameter, demonstrating the effects of perampanel at the network level. These data suggest that perampanel blocks AMPA receptors globally across the brain to account for both its antiepileptic and side-effect profile in rodents and epileptic patients.

Embryotoxicity of Psoralea corylifolia L.: in vivo and in vitro studies.

BACKGROUND: Psoralea corylifolia L. (PC) was commonly used to treat miscarriages clinically. The aim of this study was to examine its embryotoxicity in mice and embryonic stem cells (ESCs). METHODS: Quality control of PC extract including reference marker compounds, pesticide residues, and heavy metals was authenticated with HPLC, Gas chromatography-mass spectrometry (GC-MS), and inductively coupled plasma-mass spectrometry. Pregnant mice were randomly assigned into five groups and dosed with distilled water (G1), PC extract of 2 (G2), 4 (G3), or 8 g/kg/day (G4), and vitamin A (G5). Meanwhile, half maximal inhibitory concentration values for ESCs and 3T3 cells were identified in a cytotoxicity assay, and apoptosis in neuroepithelium was assessed by transmission electron microscopy. RESULTS: In the G4 group, a statistically significant decrease in the total fetus, live fetus, and gravid uterine weight, and increase in the resorbed fetus, postimplantation loss, and neuroepithelial apoptosis as well as maternal liver-weight were found (p < 0.05). CONCLUSIONS: PC extracts at 8 g/kg/day might cause fetal toxicity and maternal liver damage in mice, although it did not cause typical malformation and ESC's cytotoxicity in this experiment. Our data suggested that high dosage and long-term administration of PC preparations may not be safe for pregnant women.

Development and Characterization of Monoclonal Antibodies Against Glycophorin A Applicable for Blood Sample Processing.

The separation of plasma from blood cells is critical for the accuracy of blood tests because cellular fractions can create discrepancies in analysis. The most common method to separate blood cells from the liquid part of the blood is centrifugation, which is not always applicable in resource-constrained areas and countries. In this study, we describe the generation of monoclonal antibodies (mAbs) against glycophorin A (GPA) of human erythrocytes. BALB/c mice were immunized with human erythrocytes followed by purified GPA. The splenocytes of the immunized mice were fused with Sp2/0 myeloma cells by hybridoma technique. Hybridoma clones were screened by hemagglutination assay and enzyme-linked immunosorbent assay (ELISA). Six hybridoma clones were obtained and subcloned. The characterization of the purified mAbs demonstrates that they are able to bind and retain erythrocytes in hemagglutination assay. Furthermore, one of the mAbs 1A9 recognizes purified GPA in ELISA, whereas the other mAb 1G7 is able to immunoprecipitate GPA from human erythrocyte lysates, and a band of 38 kDa is detected. In conclusion, the anti-GPA mAbs are useful tools in developing a quick and easy way to separate blood plasma from whole blood for clinical tests, and in developing bi-specific antibodies for other clinical applications.

From gene to structure: Lactobacillus bulgaricus D-lactate dehydrogenase from yogurt as an integrated curriculum model for undergraduate molecular biology and biochemistry laboratory courses.

We have developed an integrated, project-oriented curriculum for undergraduate molecular biology and biochemistry laboratory courses spanning two semesters that is organized around the ldhA gene from the yogurt-fermenting bacterium Lactobacillus bulgaricus, which encodes the enzyme d-lactate dehydrogenase. The molecular biology module, which consists of nine experiments carried out over eleven sessions, begins with the isolation of genomic DNA from L. bulgaricus in yogurt and guides students through the process of cloning the ldhA gene into a prokaryotic expression vector, followed by mRNA isolation and characterization of recombinant gene expression levels using RT-PCR. The biochemistry module, which consists of nine experiments carried out over eight sessions, begins with overexpression of the cloned ldhA gene and guides students through the process of affinity purification, biochemical characterization of the purified LdhA protein, and analysis of enzyme kinetics using various substrates and an inhibitor, concluding with a guided inquiry investigation of structure-function relationships in the three-dimensional structure of LdhA using molecular visualization software. Students conclude by writing a paper describing their work on the project, formatted as a manuscript to be submitted for publication in a scientific journal. Overall, this curriculum, with its emphasis on experiential learning, provides hands-on training with a variety of common laboratory techniques in molecular biology and biochemistry and builds experience with the process of scientific reasoning, along with reinforcement of essential transferrable skills such as critical thinking, information literacy, and written communication, all within the framework of an extended project having the look and feel of a research experience. © 2018 by The International Union of Biochemistry and Molecular Biology, 46(3):270-278, 2018.

Iron overloaded polarizes macrophage to proinflammation phenotype through ROS/acetyl-p53 pathway.

PURPOSE: Macrophages play critical roles in inflammation and wound healing and can be divided into two subtypes: classically activated (M1) and alternatively activated (M2) macrophages. Macrophages also play important roles in regulating iron homeostasis, and intracellular iron accumulation induces M1-type macrophage polarization which provides a potential approach to tumor immunotherapy through M2 tumor-associated macrophage repolarization. However, the mechanisms underlying iron-induced M1 polarization remain unclear. METHODS: Western blotting, qRT-PCR, and flow cytometry were used to detect the polarization indexes in RAW 264.7 murine macrophages treated with iron, and Western bloting and qRT-PCR were used to detect p21 expression. The compound 2,7-dichlorofluorescein diacetate was used to measure reactive oxygen species (ROS) levels in macrophages after iron or N-acetyl-l-cysteine (NAC) treatment. The p300/CREB-binding protein (CBP) inhibitor C646 was used to inhibit p53 acetylation, and Western bloting, qRT-PCR, and immunofluorescence were used to detect p53 expression and acetylation. BALB/c mice were subcutaneously injected with H22 hepatoma cells, and macrophage polarization status was investigated after tail intravenous injection of iron. Immunohistochemical staining was used to evaluate the protein expression of cluster of differentiation 86 (CD86) and EGF-like module-containing mucin-like hormone receptor-like 1 (F4/80) in the subcutaneous tumors. RESULTS: Iron overload induced M1 polarization by increasing ROS production and inducing p53 acetylation in RAW cells, and reduction in ROS levels by NAC repressed M1 polarization and p53 acetylation. Inhibition of acetyl-p53 by a p300/CBP inhibitor prevented M1 polarization and inhibited p21 expression. These results showed that high ROS levels induced by iron overload polarized macrophages to the M1 subtype by enhancing p300/CBP acetyltransferase activity and promoting p53 acetylation.

Bioisosteres of ethyl 8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo [1,5-a][1,4]diazepine-3-carboxylate (HZ-166) as novel alpha 2,3 selective potentiators of GABAA receptors: Improved bioavailability enhances anticonvulsant efficacy.

HZ-166 has previously been characterized as an α2,3-selective GABAA receptor modulator with anticonvulsant, anxiolytic, and anti-nociceptive properties but reduced motor effects. We discovered a series of ester bioisosteres with reduced metabolic liabilities, leading to improved efficacy as anxiolytic-like compounds in rats. In the present study, we evaluated the anticonvulsant effects KRM-II-81 across several rodent models. In some models we also evaluated key structural analogs. KRM-II-81 suppressed hyper-excitation in a network of cultured cortical neurons without affecting the basal neuronal activity. KRM-II-81 was active against electroshock-induced convulsions in mice, pentylenetetrazole (PTZ)-induced convulsions in rats, elevations in PTZ-seizure thresholds, and amygdala-kindled seizures in rats with efficacies greater than that of diazepam. KRM-II-81 was also active in the 6 Hz seizure model in mice. Structural analogs of KRM-II-81 but not the ester, HZ-166, were active in all models in which they were evaluated. We further evaluated KRM-II-81 in human cortical epileptic tissue where it was found to significantly-attenuate picrotoxin- and AP-4-induced increases in firing rate across an electrode array. These molecules generally had a wider margin of separation in potencies to produce anticonvulsant effects vs. motor impairment on an inverted screen test than did diazepam. Ester bioisosters of HZ-166 are thus presented as novel agents for the potential treatment of epilepsy acting via selective positive allosteric amplification of GABAA signaling through α2/α3-containing GABA receptors. The in vivo data from the present study can serve as a guide to dosing parameters that predict engagement of central GABAA receptors.

Uptake and distribution of N, P and heavy metals in three dominant salt marsh macrophytes from Yangtze River estuary, China.

We examined the variation in aboveground biomass accumulation and tissue concentrations of nitrogen (N), phosphorus (P), copper (Cu), zinc (Zn) and lead (Pb) in Phragmites australis (common reed), Spartina alterniflora (salt cordgrass), and Scirpus mariqueter throughout the growing season (April-October 2005), in order to determine the differences in net element accumulation and distribution between the three salt marsh macrophytes in the Yangtze River estuary, China. The aboveground biomass was significantly greater in the plots of S. alterniflora than in the plots of P. australis and S. mariqueter throughout the growing season (P<0.05). In August, the peak aboveground biomass was 1246+/-89 gDW/m(2), 2759+/-250 gDW/m(2) and 548+/-54 gDW/m(2) for P. australis, S. alterniflora and S. mariqueter, respectively. The concentrations of nutrients and heavy metals in plant tissues showed similar seasonal patterns. There was a steady decline in element concentrations of the aboveground tissues from April to October. Relative element concentrations in aboveground tissues were at a peak during the spring sampling intervals with minimum levels during the fall. But the concentrations of total nitrogen and total phosphorus in the belowground tissues were relatively constant throughout growing season. Generally, trace metal concentrations in the aboveground tissues of S. mariqueter was the highest throughout the growing season, and the metal concentrations of S. alterniflora tissues (aboveground and belowground) were greater than those of P. australis. Furthermore, the aboveground pools of nutrients and metals were consistently greater for S. alterniflora than for P. australis and S. mariqueter, which suggested that the rapid replacement of native P. australis and S. mariqueter with invasive S. alterniflora would significantly improve the magnitude of nutrient cycling and bioavailability of trace metals in the salt marsh and maybe transport more toxic metals into the water column and the detrital food web in the estuary.

PTEN/MMAC1 mutations in hepatocellular carcinomas.

Mutations in the PTEN/MMAC1 gene have been identified in several types of human cancers and cancer cell lines, including brain, endometrial, prostate, breast, thyroid, and melanoma. In this study, we screened a total of 96 hepatocellular carcinoma (HCC) samples from Taiwan, where HCC is the leading cancer in males and third leading cancer in females, for mutations in the PTEN/MMAC1 gene. Complete sequence analysis of these samples demonstrated a missense mutation in exon 5 (K144I) and exon 7 (V255A) from HCC samples B6-21 and B6-2, respectively. A putative splice site mutation was also detected in intron 3 from sample B6-2. Both B6-21 and B6-2 were previously shown to contain missense mutations in the coding sequences of the p53 gene. Functional studies with the two missense mutations demonstrated that while mutation V255A in exon 7 resulted in a loss of phosphatase activity, mutation K144I in exon 5 retained its phosphatase activity. Additionally, we identified a silent mutation (P96P) in exon 5 of the PTEN/MMAC1 gene from HCC sample B6-22. These data provide the first evidence that the PTEN/MMAC1 gene is mutated in a subset of HCC samples.

Absence of PTEN germ-line mutations in men with a potential inherited predisposition to prostate cancer.

Epidemiological studies have demonstrated that men with a family history of prostate cancer are at an increased risk for this disease. This important observation has led a number of research teams, including our own, to collect DNA samples and clinical data from prostate cancer families, with the goal of localizing and characterizing prostate cancer susceptibility genes. The candidate tumor suppressor gene PTEN (also called MMAC1) has recently been shown to be somatically altered in several common malignancies, including cancers of the brain, kidney, skin, thyroid, endometrium, breast, and prostate. Germ-line mutations in this gene, which maps to chromosome 10q23, have been associated with Cowden disease, an autosomal dominant cancer predisposition syndrome that is characterized by multiple hamartomas. Although prostate cancer is not typically associated with Cowden disease, previous studies of sporadic prostate cancers demonstrate loss of heterozygosity at 10q23 loci in approximately 25% of cases. We, therefore, hypothesized that germ-line mutations in the PTEN gene may predispose to prostate cancer in a subset of families, particularly those in which cancers of the breast, kidney, and/or thyroid also segregate. To test this hypothesis, DNA was isolated from whole blood of 11 prostate cancer patients from 10 unrelated families. Four of the 10 families met the previously established clinical criteria for hereditary prostate cancer. Eight of the II men had at least one second primary malignancy, including cases of neuroendocrine cancer, glioblastoma multiforme, melanoma, kidney, and thyroid cancer. Although we identified some common as well as some unique polymorphisms, no nonsense or missense mutations were identified in any of the 11 samples. To further examine the possibility that PTEN mutations contribute to prostate cancer predisposition, we also studied the probands from each of 10 families with early-onset and/or multiple individuals with prostate cancer. Sequence analysis of the PTEN gene in these 10 men also revealed no mutations or novel polymorphisms. We conclude that germ-line mutations in the PTEN are unlikely to contribute in a significant way to the inherited predisposition to prostate cancer.

The utility of MRI for pre-operative T and N staging of gastric carcinoma: a systematic review and meta-analysis.

OBJECTIVE: To perform a meta-analysis and literature review regarding the diagnostic accuracy of MRI for pre-operative tumour depth invasion (T) and regional lymph node invasion (N) staging of gastric carcinoma (GC). METHODS: Articles were identified through systematic search of Medline, PubMed, Cochrane Library, Web of Science, Springerlink and several Chinese databases. The study quality was assessed by the quality assessment for studies of diagnostic accuracy. 2 reviewers independently extracted and assessed the data from 11 eligible studies. A meta-analysis was then carried out. Subgroup and sensitivity analyses were also performed. RESULTS: 11 studies (439 patients) were finally included in the current review. Among these studies, the significant evidence of heterogeneity was only discovered for specificity in T4 stage (I(2) = 59.8%). Pooled sensitivity and specificity of MRI to diagnose T stage tumour (T3-4 vs T1-2) were 0.93 [95% confidence interval (CI), 0.89-0.96] and 0.91 (95% CI, 0.87-0.95), respectively. Pooled estimates of sensitivity and specificity of MRI to diagnose N stage tumour (N0 vs N+) were 0.86 (95% CI, 0.80-0.92) and 0.67 (95% CI, 0.54-0.79), respectively. Subgroup analyses showed that diffusion-weighted imaging was more helpful for T staging. CONCLUSION: The present systematic review suggests that MRI has a good diagnostic accuracy for pre-operative T staging of GC and should be widely used in clinical work. However, the ability for N staging is relatively poor on MRI. ADVANCES IN KNOWLEDGE: In the pre-operative staging of GC, MRI was a useful tool and may enhance accuracy for the T staging of advanced GC.

PTCH mutations in squamous cell carcinoma of the skin.

Ultraviolet light exposure is the major risk factor for the development of squamous cell carcinoma in Caucasians. Mutations in the tumor suppressor gene p53 have been identified in both squamous cell carcinomas and basal cell carcinomas. The human homolog of the Drosophila patched gene, has been shown to be mutated in sporadic basal cell carcinomas; however, mutations in the patched gene have not been found in squamous cell carcinoma. In this study, we screened a total of 20 squamous cell carcinoma samples for mutations in the patched gene. Using polymerase chain reaction-single strand conformation polymorphism as an initial screening method, we identified one non-sense mutation, two mis-sense mutations and three silent mutations in five squamous cell carcinoma samples. In one squamous cell carcinoma sample, we identified a tandem GG-->AA transitional change at nucleotide 3152 in exon 18 of the patched gene that resulted in a premature stop codon at codon 1051. The three squamous cell carcinoma samples containing non-sense and mis-sense mutations were isolated from individuals with histories of multiple basal cell carcinoma. Sequence analysis of the p53 gene in these five squamous cell carcinoma samples identified one CC-->TT and three C-->T ultraviolet-specific nucleotide changes. Our study provides evidence that the patched gene is mutated in squamous cell carcinoma from individuals with a history of multiple basal cell carcinoma. The identification of ultraviolet-specific nucleotide changes in both tumor suppressor genes supports the notion that ultraviolet exposure plays an important part in the development of squamous cell carcinoma.

Chinese village women as visual anthropologists: a participatory approach to reaching policymakers.

In developing countries, rural women are often neither seen nor heard, despite their extraordinary contribution to the labor force. Photo novella is an innovative methodology that puts cameras in the hands of rural women and other constituents who seldom have access to those who make decisions over their lives. As an educational tool, the practice of photo novella has three main goals: (1) to empower rural women to record and reflect their lives, especially health needs, from their own point of view; (2) to increase their collective knowledge about women's health status; and (3) to inform policymakers and the broader society about health and community issues that are of greatest concern to rural women. In this paper we analyze the third goal: the contributions and limitations of photo novella as a tool for informing policymakers. We conceptualize first the theoretical and practical underpinnings of photo novella. After tracing the relationships among empowerment education, feminist theory, documentary photography and policy, we describe photo novella within the broader context of the Ford Foundation-supported Yunnan Women's Health and Development Program and explain its application for influencing policy based on our experience carrying out photo novella in China.

Mode of action of anhydrofulvic acid against Candida utilis ATCC 42402 under acidic condition.

The mode of action of anhydrofulvic acid against Candida utilis ATCC 42402 was investigated under acidic conditions. Anhydrofulvic acid inhibited the incorporation of radioactive precursors into DNA, RNA, protein and lipid fractions. Although it did not induce leakage of intracellular materials from the treated cells, it had inhibitory effects on both endogenous and exogenous cellular respiration. Moreover, it inhibited mitochondrial respiration of Candida utilis ATCC 42402 using both succinate and cytochrome c as respiratory substrates, but not using NADH. Unexpectedly, the inhibition against isolated mitochondria was observed at pH 7.0. These results suggested that the action site against the respiratory inhibition of anhydrofulvic acid might be involved in succinate dehydrogenase, complex II in the mitochondrial electron transport chain of the yeast cells. Judging from the inhibitory effect of anhydrofulvic acid on mitochondria detected at pH 7.0, it was postulated that the antifungal activity at a low pH level might depend on the elevation of drug permeability to the cell membrane under acidic conditions.

The mode of action of AKD-2C, an antifungal antibiotic from Streptomyces sp. OCU-42815.

The mechanism of the antifungal action of AKD-2C was studied by using Torulaspora delbrueckii IFO 1621 as a model. AKD-2C slightly inhibited the incorporation of radioactive precursors into protein, RNA and lipid, but not into DNA. On the other hand, AKD-2C greatly enhanced the leakage of K+ ions from treated cells and showed a potent effect on liposomal glucose leakage. Using electron microscopic studies, though drastic morphological changes were not observed, an increase in cell membrane irregularities and swelling of the mitochondria caused by AKD-2C were demonstrated. These results suggest that the antifungal action of AKD-2C is due to effects on the yeast cell membrane.

L-2,5-dihydrophenylalanine, an inducer of cathepsin-dependent apoptosis in human promyelocytic leukemia cells (HL-60).

L-2,5-Dihydrophenylalanine (DHPA), a phenylalanine analogue, induced apoptosis in human promyelocytic leukemia cells (HL-60). This apoptosis was demonstrated by morphological changes of the cells, such as fragmentation of nuclei and chromatin condensation, and by some evidence found in biochemical analysis, such as DNA ladder and activation of caspase 3. The DHPA-induced apoptosis was prevented by a pan-caspase inhibitor, Z-VAD-fmk, and a cysteine protease inhibitor, E-64d, which inhibits calpains and cathepsin B and L. A calpain inhibitor, Z-LL-H, did not affect this apoptosis. A cathepsin B specific inhibitor, CA074-Me, prevented only chromatin condensation. However, E-64d and a cathepsin L specific inhibitor, Z-FY(t-Bu)-dmk, protected the cells from both chromatin condensation and oligonucleosomal DNA fragmentation. As proceeding to the apoptotic process, the activities of both cathepsin B and L increased gradually. These results indicated that DHPA was an inducer of cathepsin-dependent apoptosis in HL-60 cells.

AKD-2A, B, C and D, new antibiotics from Streptomyces sp. OCU-42815. Taxonomy, fermentation, isolation, structure elucidation and biological activity.

An antibiotic complex, AKD-2, was isolated from the mycelial cake of Streptomyces sp. OCU-42815. The lipophilic substances in this complex were further purified by a recycling HPLC procedure and were designated AKD-2A, C and D. AKD-2B was obtained as a mixture of AKD-2B1 and AKD-2B2. These substances were identified as monoglycerides having branched chain fatty acids and exhibited both antibacterial and antifungal activities.

Evaluation of the biocompatibility of two new diamond-like stent coatings (Dylyn) in a porcine coronary stent model.

UNLABELLED: Hydrogenated diamond-like carbon films (DLC, a-C:H), deposited using plasma-assisted or ion beam-assisted techniques, offer great potential as self-lubricating coatings in many tribological applications. Additionally, studies on biocompatibility have shown that DLC is an inert, impervious hydrocarbon with properties suitable for use in the biomedical field. One particular class of modified DLC coatings are diamond-like nanocomposite coatings (DLN or Dylyn , Bekaert, Kortrijk, Belgium), which offer promising solutions for many industrial applications. In this study, the biocompatibility of two diamond-like stent coatings are evaluated in a porcine coronary stent model. METHODS: Either coated or non-coated stents were randomly implanted in two coronary arteries of 20 pigs so that each group contained 13 stented arteries. Pigs underwent a control angiogram at 6 weeks and were then sacrificed. Quantitative coronary analysis before, immediately after stent implantation, and at 6 weeks was performed using the semi-automated Polytron 1000 system (Siemens, Erlangen, Germany). Morphometry was performed using a computerized morphometric program. Angiographic analysis showed similar baseline selected arteries and post-stenting diameters. At 6-week follow-up, there was no significant difference in minimal stent diameter. Histopathology revealed a similar injury score in the 3 groups. Inflammation was significantly increased in the DLN-DLC coating group. Thrombus formation was significantly decreased in both coated stent groups. Neointimal hyperplasia was decreased in both coated stent groups; however, the difference with the non-coated stents was not statistically significant. Area stenosis was lower in the DLN-coated stent group than in the control group (41 +/- 17% vs. 54 +/- 15%; p = 0.06). CONCLUSION: The results indicate that the diamond-like nanocomposite stent coatings are compatible, resulting in decreased thrombogenicity and decreased neointimal hyperplasia. Covering this coating with another diamond-like carbon film (DLC) resulted in an increased inflammatory reaction and no additional advantage compared to the single-layer diamond-like nanocomposite coating.

Synergistic anti-tumor effect of ultrasound and hematoporphyrin on sarcoma180 cells with special reference to the changes of morphology and cytochrome oxidase activity of tumor cells.

This study is aimed at evaluating the inhibitory effects of the association of hematoporphyrin and ultrasound at variable intensities with a fixed frequency of 1.1MHz in tumor nodules. Specifically, the effects were studied both in solid and ascitic S180 tumors transplanted in mice by clinical, cytochemical and ultrastructural evaluation. The results indicated that the use of hematoporphyrin alone had no significant effect on destroying tumor cells. The ultrasound alone had little effect. Interestingly, the inhibition was much more effective when hematoporphyrin was combined with ultrasound. The inhibition was 3 times better than ultrasound alone and 8 times better than hematoporphyrin used alone. Our results also indicated that the changes on cell structure and cytochrome oxidation activity are important factors that could inhibit tumor cell growth and induce cell death. Apoptosis of tumor cells could be induced by hematoporphyrin. Our study investigated the killing mechanism on S180 tumor cells by using hematoporphyrin and low frequency ultrasound at cell, tissue and individual level.

Antifungal activity of radicicol against Mucor flavus IFO 9560.

The antifungal activity of radicicol against Mucor flavus IFO 9560 was investigated. Radicicol induced bursting of spores during germination and morphological changes of the mycelial tip such as overbranching and swelling during exponential growth. In addition, radicicol showed a dose-dependent inhibitory effect on spore germination. Radicicol also inhibited the incorporation of radioactive precursors into DNA, RNA, protein, and chitin fractions by 20-30%, but not into the lipid fraction. There were no inhibitory effects on either endogenous or exogenous cellular respiration. Moreover, leakage of UV-absorbing, phenol sulfate-positive, or folin reagent-positive materials from the mycelia was not observed at an early stage of growth inhibition. On the other hand, kinetic studies of chitin synthase in the untreated mycelia revealed that radicicol noncompetitively inhibited the enzyme at Ki of 87 microM. Furthermore, upon incubation of the normal mycelia with radicicol in 50 mM KH2PO4-NaOH buffer (pH 6.5) containing 10 mM MgCl2, chitin synthase from the mycelia was inactivated gradually at first, and completely after 24-h incubation. These results suggested that radicicol exhibits the antifungal activity by disturbing cell wall biosynthesis through the inactivation of chitin synthase. However, at an early stage of growth inhibition, radicicol was thought to affect cellular function including nucleic acid and protein syntheses, in addition to the reversible noncompetitive inhibition of chitin synthase.