In Vitro Evaluation of the Inhibitory Potential of Pharmaceutical Excipients on Human Carboxylesterase 1A and 2

Two major forms of human carboxylesterase (CES), CES1A and CES2, dominate the pharmacokinetics of most prodrugs such as imidapril and irinotecan (CPT-11). Excipients, largely used as insert vehicles in formulation, have been recently reported to affect drug enzyme activity. The influence of excipients on the activity of CES remains undefined. In this study, the inhibitory effects of 25 excipients on the activities of CES1A1 and CES2 were evaluated. Imidapril and CPT-11 were used as substrates and cultured with liver microsomes in vitro. Imidapril hydrolase activities of recombinant CES1A1 and human liver microsomes (HLM) were strongly inhibited by sodium lauryl sulphate (SLS) and polyoxyl 40 hydrogenated castor oil (RH40) [Inhibition constant (K_i) = 0.04±0.01 μg/ml and 0.20±0.09 μg/ml for CES1A1, and 0.12±0.03 μg/ml and 0.76±0.33 μg/ml, respectively, for HLM]. The enzyme hydrolase activity of recombinant CES2 was substantially inhibited by Tween 20 and polyoxyl 35 castor oil (EL35) (K_i = 0.93±0.36 μg/ml and 4.4±1.24 μg/ml, respectively). Thus, these results demonstrate that surfactants such as SLS, RH40, Tween 20 and EL35 may attenuate the CES activity; such inhibition should be taken into consideration during drug administration.     

Hydrogel-Forming Microneedle Arrays Allow Detection of Drugs and Glucose In Vivo: Potential for Use in Diagnosis and Therapeutic Drug Monitoring

We describe, for the first time the use of hydrogel-forming microneedle (MN) arrays for minimally-invasive extraction and quantification of drug substances and glucose from skin in vitro and in vivo. MN prepared from aqueous blends of hydrolysed poly(methyl-vinylether-co-maleic anhydride) (11.1% w/w) and poly(ethyleneglycol) 10,000 daltons (5.6% w/w) and crosslinked by esterification swelled upon skin insertion by uptake of fluid. Post-removal, theophylline and caffeine were extracted from MN and determined using HPLC, with glucose quantified using a proprietary kit. In vitro studies using excised neonatal porcine skin bathed on the underside by physiologically-relevant analyte concentrations showed rapid (5 min) analyte uptake. For example, mean concentrations of 0.16 μg/mL and 0.85 μg/mL, respectively, were detected for the lowest (5 μg/mL) and highest (35 μg/mL) Franz cell concentrations of theophylline after 5 min insertion. A mean concentration of 0.10 μg/mL was obtained by extraction of MN inserted for 5 min into skin bathed with 5 μg/mL caffeine, while the mean concentration obtained by extraction of MN inserted into skin bathed with 15 μg/mL caffeine was 0.33 μg/mL. The mean detected glucose concentration after 5 min insertion into skin bathed with 4 mmol/L was 19.46 nmol/L. The highest theophylline concentration detected following extraction from a hydrogel-forming MN inserted for 1 h into the skin of a rat dosed orally with 10 mg/kg was of 0.363 μg/mL, whilst a maximum concentration of 0.063 μg/mL was detected following extraction from a MN inserted for 1 h into the skin of a rat dosed with 5 mg/kg theophylline. In human volunteers, the highest mean concentration of caffeine detected using MN was 91.31 μg/mL over the period from 1 to 2 h post-consumption of 100 mg Proplus^® tablets. The highest mean blood glucose level was 7.89 nmol/L detected 1 h following ingestion of 75 g of glucose, while the highest mean glucose concentration extracted from MN was 4.29 nmol/L, detected after 3 hours skin insertion in human volunteers. Whilst not directly correlated, concentrations extracted from MN were clearly indicative of trends in blood in both rats and human volunteers. This work strongly illustrates the potential of hydrogel-forming MN in minimally-invasive patient monitoring and diagnosis. Further studies are now ongoing to reduce clinical insertion times and develop mathematical algorithms enabling determination of blood levels directly from MN measurements.     

The development of isocitric lyase activity in germinating cotton seed

In cotyledons of germinating cotton (Gossypium hirsutum L. var. Stoneville 213) seedlings, in the dark, isocitric lyase (EC 4.1.3.1) activity peaks after 2 days and thereafter slowly declines to a negligible value after 8 days. The maximum activity of this enzyme in cotyledons of 2-day-old seedlings was 16.2 μmoles of glyoxylate formed/15 min·10 cotyledon pairs. Actinomycin D at a concentration of 10 μg/ml, if added to the imbibing solution, completely prevents the development of isocitric lyase activity in these germinating seed. In cotyledons of germinating cotton seedlings, in the light, isocitric lyase activity peaks after 2 to 3 days and sharply declines to a negligible value after 4 days. The maximum activity of this enzyme in cotyledons of 2- to 3-day-old seedlings was 13.2 μmoles of glyoxylate formed/15 min·10 cotyledon pairs. Actinomycin D at a concentration of 10 μg/ml, if added to the imbibing solution, severely inhibits the development of enzyme activity.     

In Vitro Antimicrobial Activity and Human Pharmacology of Cephalexin, a New Orally Absorbed Cephalosporin C Antibiotic

Concentrations of cephalexin (an orally absorbed derivative of cephalosporin C) in serum and urine were determined in normal volunteers and patients. The in vitro antibacterial activity was also studied. All strains of group A β-hemolytic streptococci and Diplococcus pneumoniae were inhibited by 3.1 μg/ml. Of the Staphylococcus aureus strains, 88% were inhibited by 6.3 μg/ml, and 12.5 μg/ml was inhibitory for all S. aureus, 80% of Escherichia coli, 72% of Klebsiella-Aerobacter, and 56% of Proteus mirabilis strains. About 90 to 96% of E. coli, Klebsiella Aerobacter, and P. mirabilis strains were inhibited by 25 μg of cephalexin per ml. Pseudomonas and indole-positive Proteus strains proved to be quite resistant to cephalexin. Cephalexin was well absorbed after oral administration. A peak serum concentration of cephalexin of at least 5 μg/ml was achieved in each volunteer with 250 and 500-mg doses. A mean peak serum concentration of 7.7 μg/ml was achieved with 250-mg doses; 12.3μg/ml was achieved with 500-mg doses of antibiotic. Food did not interfere with absorption. Probenecid enhanced both the peak serum concentration and the duration of antibiotic activity in the serum. Over 90% of the administered dose was excreted in the urine within 6 hr. The mean peak serum concentration of cephalexin after an oral dose of 500 mg was adequate to inhibit all group A streptococci, D. pneumoniae, and S. aureus, 85% of E. coli, and about 40 to 75% of Klebsiella-Aerobacter and P. mirabilis strains. Levels of cephalexin in urine were adequate to inhibit over 90% of E. coli, and P. mirabilis and 80 to 96% of Klebsiella-Aerobacter strains.     

Screening of ethnic medicinal plants of South India against influenza (H1N1) and their antioxidant activity

Antiviral activity against H1N1 influenza was studied using ethnic medicinal plants of South India. Results revealed that Wrightia tinctoria (2.25 μg/ml) was one of the best antidotes against H1N1 virus in terms of inhibitory concentration of 50% (IC₅₀) whereas the control drug Oseltamivir showed 6.44 μg/ml. Strychnos minor, Diotacanthus albiflorus and Cayratia pedata showed low cytotoxicity (>100) to the MDCK (Malin darby canine kidney) cells by cytotoxicity concentration of 50% (CC₅₀) and possessed antiviral activity suggesting that these plants can be used as herbal capsules for H1N1 virus. W. tinctoria and S. minor showed high therapeutic indexes (TI) such as 12.67 and 21.97 suggesting that those plants can be used for anti-viral drug development. The CC₅₀ values of Eugenia singampattiana (0.3 μg/ml), Vitex altissima (42 μg/ml), Salacia oblonga (7.32 μg/ml) and Salacia reticulata (7.36 μg/ml) resulted in cytotoxicity of the MDCK cells, due to their high phenolic content. Findings from this study state that the plant W. tinctoria can be a potent source for third generation anti-viral drug development against H1N1.     

Angiotensin I-Converting-Enzyme-Inhibitory and Antibacterial Peptides from Lactobacillus helveticus PR4 Proteinase-Hydrolyzed Caseins of Milk from Six Species

Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine α_S1-casein (α_S1-CN) 24-47 fragment (f24-47), f169-193, and β-CN f58-76; ovine α_S1-CN f1-6 and α_S2-CN f182-185 and f186-188; caprine β-CN f58-65 and α_S2-CN f182-187; buffalo β-CN f58-66; and a mixture of three tripeptides originating from human β-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (S)-N-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 μg/ml (100 μmol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC₅₀) of some of the crude peptide fractions was very low (16 to 100 μg/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC₅₀s were confirmed. An antibacterial peptide corresponding to β-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as Enterococcus faecium, Bacillus megaterium, Escherichia coli, Listeria innocua, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. The MIC for E. coli F19 was ca. 50 μg/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of L. helveticus PR4 or by trypsin and chymotrypsin.     

Bioactive endophytic fungi isolated from Caesalpinia echinata Lam. (Brazilwood) and identification of beauvericin as a trypanocidal metabolite from Fusarium sp.

Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro assays. The organic extracts from three isolates showed antibacterial activity against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration (MIC) 32-64 μg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC 64 μg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 μg/mL), Candida albicans and Candida tropicalis (MIC 64-128 μg/mL). Fourteen extracts at a concentration of 20 μg/mL showed antitumour activities against human breast cancer and human renal cancer cells, while two isolates showed anti-tumour activities against human melanoma cancer cells. Six extracts were able to reduce the proliferation of human peripheral blood mononuclear cells, indicating some degree of selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania) amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 μg/mL. The trypanocidal extract obtained from Fusarium sp. [{"type":"entrez-nucleotide","attrs":{"text":"KF611679","term_id":"560187972","term_text":"KF611679"}}KF611679] culture was subjected to bioguided fractionation, which revealed beauvericin as the compound responsible for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a half maximal inhibitory concentration of 1.9 μg/mL (2.43 μM) in a T. cruzi cellular culture assay.     

Effect of Vancomycin on the Growth of Psittacosis-Trachoma Agents Cultivated in Eggs and Cell Culture

The antibiotic vancomycin was effective in preventing bacterial contamination during studies with psittacosis and trachoma (PT) agents. This antibiotic was not toxic to chick embryos at 80 mg per egg, or to HeLa 229 cells cultivated in a medium containing 3,200 μg/ml of vancomycin; however, it was toxic to LLC-MK2 cells at a concentration of 1,600 μg/ml. Vancomycin did not affect the growth of a variety of PT agents at a concentration of 2 mg per egg or 800 μg per ml of cell culture medium, but it did inhibit the growth of common gram-positive bacterial air contaminants.     

Extracellular ribonuclease formation in Bacillus subtilis and its stimulation by actinomycin D

1. Extracellular ribonuclease is produced linearly for at least 3hr. by washed post-logarithmic-phase cells of Bacillus subtilis suspended in a medium containing maltose (1%) and casein hydrolysate (0·5%). 2. Low concentrations of actinomycin D (less than 2μg./ml.) stimulate ribonuclease formation, the maximum effect being observed with a concentration of 1μg./ml. Concentrations greater than 2μg./ml. are inhibitory. There is no parallel stimulation of α-amylase formed under the same conditions, and [¹⁴C]uracil incorporation into a perchloric acid-insoluble form is inhibited. 3. The actinomycin D-induced stimulation is not due to the presence of an activator, nor is the inhibition due to the release of an inhibitor by the cells. The effect is on the amount of ribonuclease produced in the medium. 4. Extracellular ribonuclease formation is partially inhibited by anaerobiosis, 2,4-dinitrophenol, sodium azide and by chloramphenicol and puromycin. 5. High concentrations of antibiotic do not completely inhibit ribonuclease formation, but a basal amount of enzyme representing 20min. synthesis in an uninhibited system is always produced. This `antibiotic-insensitive' enzyme could possibly represent preformed enzyme `in the pipe-line' en route to secretion. 6. The stimulated appearance of ribonuclease in the presence of 1μg. of actinomycin D/ml. is shown to be dependent on enzyme synthesis. The mechanism of this effect is discussed.     

In Vitro Susceptibility of Neisseria gonorrhoeae to Nine Antimicrobial Agents

The in vitro action of nine antibiotics was tested by the agar streak method against 45 gonococcal strains isolated from penicillin-therapy failures. The penicillin susceptibility range of these strains was 0.003 to 1.32 μg/ml, and the tetracycline susceptibility range was 0.125 to 2.0 μg/ml. Minimal inhibitory concentrations of minocycline and doxycycline paralleled the activity of tetracycline and ranged from 0.125 to 1.0 μg/ml and 0.125 to 2.0 μg/ml, respectively. Rifampicin, with a narrow range of 0.5 to 1.0 μg/ml, inhibited 75% of the strains at 0.5 μg/ml. The range for cephaloridine and cephaloglycine was 0.5 to 20.0 μg/ml, but another cephalosporium derivative, cephalexin, exhibited greater activity in its range of 0.25 to 20.0 μg/ml. A semisynthetic penicillin, carbenicillin, with a range of 0.025 to 0.75 μg/ml, displayed more activity against the lower susceptible penicillin G gonococcal strains.     

Extraction of saponins and toxicological profile of Teucrium stocksianum boiss extracts collected from District Swat,Pakistan

Background

The current era is facing challenges in the management of neoplasia and weeds control. The currently available anti-cancer and herbicidal drugs are associated with some serious side effects. Therefore numerous researchers are trying to discover and develop plant based alternative particularly for the rational management of cancer and weed control. Teucrium stocksianum possess antioxidant and analgesic activities. The current study was designed to evaluate crude saponins (CS), methanolic extract and sub-fractions of T. stocksianum for cytotoxic and phytotoxic potentials. CS, methanolic extract and sub-fractions were extracted from powdered plant material using different solvents. Cytotoxic potential of the extracts at a dose of 10, 100 and 1000 μg/ml were evaluated against Brine shrimp’s nauplii. Phytotoxic assay also performed at the same concentration against Lemna minor. Etoposide and Paraquat were used as positive controls in cytotoxic and phytotoxic assays respectively.

Results

The percent yield of crude saponins was (5%). CS demonstrated tremendous brine shrimp lethality showing < 10 μg/ml LC₅₀. The n-hexane (HF) and chloroform fractions (CF) demonstrated excellent cytotoxicity with 80 and 55 μg/ml LC₅₀ respectively. Whereas the methanolic extract (TSME), ethyl acetate (EAF) and aqueous fractions (AF) revealed moderate cytotoxicity showing 620, 860 and 1000 μg/ml LC₅₀ values respectively. In phytotoxic assay profound inhibition was displayed by HF (96.67%) and TSME (95.56%, 30 μg/ml LC₅₀) against the growth of Lemna minor at 1000 μg/ml respectively. Both CF and EAF demonstrated profound phytoxicity (93.33%) respectively at highest concentration (1000 μg/ml), while AF and CS demonstrated weak phytotoxicity with 1350 and 710 μg/ml LC₅₀ values respectively.

Conclusion

Cytotoxicity and phytotoxicity assays indicated that the crude saponins, n-hexane and chloroform fractions of T. stocksianum could play a vital role in the treatment of neoplasia and as potential natural herbicides. Therefore these sub-fractions are recommended for further investigation with the aim to isolate novel anti-cancer and herbicidal compounds.     

Aqueous and Alcoholic Extracts of Triphala and Their Active Compounds Chebulagic Acid and Chebulinic Acid Prevented Epithelial to Mesenchymal Transition in Retinal Pigment Epithelial Cells,by Inhibiting SMAD-3 Phosphorylation

Epithelial to Mesenchymal Transition (EMT) of the retinal pigment epithelium is involved in the pathogenesis of proliferative vitreoretinopathy (PVR) that often leads to retinal detachment. In this study, Triphala, an ayurvedic formulation and two of its active ingredients, namely chebulagic acid and chebulinic acid were evaluated for anti-EMT properties based on in vitro experiments in human retinal pigment epithelial cell line (ARPE-19) under TGFβ1 induced conditions. ARPE-19 cells were treated with TGFβ1 alone or co-treated with various concentrations of aqueous extract (AqE) (30 - 300 μg/ml); alcoholic extract (AlE) (50 - 500 μg/ml) of triphala and the active principles chebulagic acid (CA) and chebulinic acid (CI) (CA,CI: 50 - 200 μM). The expression of EMT markers namely MMP-2, αSMA, vimentin and the tight junction protein ZO-1 were evaluated by qPCR, western blot and immunofluorescence. The functional implications of EMT, namely migration and proliferation of cells were assessed by proliferation assay, scratch assay and transwell migration assay. AqE, AlE, CA and CI reduced the expression and activity of MMP-2 at an ED50 value of 100 μg/ml, 50 μg/ml, 100 μM and 100 μM, respectively. At these concentrations, a significant down-regulation of the expression of αSMA, vimentin and up-regulation of the expression of ZO-1 altered by TGFβ1 were observed. These concentrations also inhibited proliferation and migration of ARPE-19 cells induced by TGFβ1. EMT was found to be induced in ARPE-19 cells, through SMAD-3 phosphorylation and it was inhibited by AqE, AlE, CA and CI. Further studies in experimental animals are required to attribute therapeutic potential of these extracts and their active compounds, as an adjuvant therapy in the disease management of PVR.     

Fecal Calprotectin Concentrations in Healthy Children Aged 1-18 Months

Objective

Fecal calprotectin (FC) is an established biomarker of gut inflammation. The aim of this study was to evaluate FC concentrations in healthy children between 1 and 18 months of age.

Methods

Healthy children aged 1-18 months were enrolled in this study at the Department of Children''s Health Care in Shanghai, China. Children’s stool samples were collected and analyzed, and FC concentration was determined using a commercially available enzyme-linked immunosorbent assay (ELISA). The children''s weights and lengths were measured. Parents were asked to complete a brief questionnaire regarding several clinical and sociodemographic factors.

Results

The FC concentrations were unevenly distributed; the median FC concentration was 174.3 μg/g (range: 6.0-1097.7 μg/g) or 2.241 log10 μg/g (range: 0.775-3.041 log10 μg/g) for all 288 children. The children were divided into several age groups: 1-3 months, 3-6 months, 6-9 months, 9-12 months and 12-18 months. The median FC concentrations for these age groups were 375.2 μg/g (2.574 log10 μg/g), 217.9 μg/g (2.338 log10 μg/g), 127.7 μg/g (2.106 log10 μg/g), 96.1 μg/g (1.983 log10 μg/g) and 104.2 μg/g (2.016 log10 μg/g), respectively. A significant correlation between age and FC concentration was found (r=-0.490, p<0.001). A simple correlation analysis of weight-for-length Z-scores or weight-for-age Z-scores vs. FC concentrations showed that these variables were negatively correlated (Spearman’s rho=-0.287, p<0.001; Spearman’s rho=-0.243, p<0.001, respectively).

Conclusions

The FC levels of children aged 1-18 months exhibit a downward trend with increasing age and are greater than the normal levels observed in healthy adults. In healthy children aged <6 months, FC levels are high. In children aged 6-18 months, FC concentrations are relatively low but are still higher than those of children aged >4 years.     

Fecal Calprotectin in Healthy Children Aged 1-4 Years

Objective

Calprotectin has been well emulated recently in adults as well as in children. The aim of this study was to assess fecal calprotectin concentrations in healthy children aged from 1 to 4 years.

Methods

Volunteers were enlisted from 3 nurseries. A brief questionnaire was used to ensure these children meet the inclusion criteria, and some clinical and sociodemographic factors were collected. Anthro software (version 3.1) was used to calculated Length-for-age Z-scores (LAZ), weight-for-age Z-scores (WAZ), and weight-for-length Z-scores (WLZ) respectively. Fecal calprotectin was detected by a commercially available ELISA.

Results

In total 274 children were recruited, with age ranging from 1 to 4 years old. The median FC concentration was 83.19 μg/g [range 4.58 to 702.50 μg/g, interquartile range (IQR) 14.69–419.45 μg/g] or 1.92 log10 μg/g (range 0.66 log10 to 2.85 log10 μg/g, IQR 1.17 log10-2.62 log10 μg/g). All of the children were divided into three groups, 1–2 years (12–24 months), 2–3 years (24–36 months), 3–4 years (36–48 months), with median FC concentrations 96.14 μg/g (1.98 log10 μg/g), 81.48 μg/g (1.91 log10 μg/g), 65.36 μg/g (1.82 log10 μg/g), respectively. There was similar FC level between boys and girls. FC concentrations showed a downward trend by the growing age groups. A statistic difference was found in FC concentrations among groups 1–2 years, 2–3 years and 3–4 years (P = 0.016). In inter-groups comparison, a significant difference was found between children aged 1–2 years and children aged 3–4 years (P = 0.007). A negative correlation trend was found between age and FC concentration (Spearman''s rho = -0.167, P = 0.005) in all the participants. A simple correlation was performed among WLZ, WAZ, birth weight, or birth length with FC, and there was no correlation being observed.

Conclusion

Children aged from 1 to 4 years old have lower FC concentrations compared with healthy infants (<1years), and higher FC concentrations when comparing with children older than 4 years and adults.     

Fusarium poae and Fusarium crookwellense, Fungi Responsible for the Natural Occurrence of Nivalenol in Hokkaido

To determine the reasons for the natural occurrence of nivalenol in the northernmost area of Japan, scabby wheat was harvested from 19 crop fields in Hokkaido. Mycological surveys and analysis for mycotoxin contamination were performed. Among 13 wheat grain samples harvested in seven locations, 9, 2, and 6 samples were positive for deoxynivalenol, nivalenol, and zearalenone, respectively, at levels ranging from 0.03 to 1.28 μg/g, 0.04 to 1.22 μg/g, and 2 to 25 ng/g, respectively. The predominant Fusarium species of the scabby wheat examined were F. sporotrichioides, F. avenaceum, F. poae, and F. crookwellense. Fifteen of 48 F. poae isolates and all four F. crookwellense isolates were screened for the production of seven derivatives of trichothecenes and zearalenone respectively, on rice culture. One isolate of F. poae produced diacetoxyscirpenol alone (4.3 μg/g); seven produced nivalenol (1.3 to 23.8 μg/g), 4-acetylnivalenol (0.1 to 4.6 μg/g), and diacetoxyscirpenol (0.9 to 99.5 μg/g); and five produced nivalenol alone (0.4 to 3.5 μg/g). The remaining two isolates produced no trichothecenes. Zearalenone production was not found in any isolate of F. poae tested. All isolates of F. crookwellense produced nivalenol (0.9 to 22.5 μg/g), 4-acetylnivalenol (0.5 to 25.0 μg/g), and zearalenone (1.4 to 162.5 μg/g). From these results, it is apparent that deoxynivalenol and zearalenone, and occasionally nivalenol, occur naturally throughout Hokkaido, and it is suggested that nivalenol-producing F. poae and F. crookwellense strains are responsible for the natural contamination with nivalenol found in the northernmost area of Japan. Furthermore, it was found for the first time that several isolates of F. poae distributed in Hokkaido possessed the ability to produce both type A and type B trichothecenes.     

On the regulation of tyrosine transaminase, glutamatic dehydrogenase and aspartic transaminase in Tetrahymena

Aspartic transaminase, tyrosine transaminase, lactic dehydrogenase, and glutamic dehydrogenase were studied in Tetrahymena pyriformis in order to gain a better understanding of the control of the entrance and exit of metabolic intermediates to and from the major carbohydrate pathways. Glucose decreased the activity of aspartic transaminase, tyrosine transaminase and glutamic dehydrogenase but not lactic dehydrogenase. Actinomycin D (6 and 12 μg/ml) blocked the decrease in glutamic dehydrogenase and aspartic transaminase activity caused by glucose; 12 μg/ml partially prevented the decrease in tyrosine transaminase activity. Actinomycin D alone had little effect on enzyme activity. Uracil incorporation into RNA was doubled by 6 μg/ml actinomycin D, a concentration which did not alter the RNA content of the cells. At 12 μg/ml this drug caused a small decrease in RNA spec. act. Cycloheximide at 10 μg/ml, a concentration which inhibited protein synthesis by 70%, caused a three-fold increase in aspartic transaminase and a two-fold increase in glutamic dehydrogenase. In the presence of both cycloheximide and glucose, the drug effect predominated. Thus both actinomycin D and cycloheximide blocked the glucose-induced decrease in enzyme activity. These results suggest that the levels of aspartic transaminase, glutamic dehydrogenase, and probably tyrosine transaminase are regulated at least in part by a degradative control system.     

In Vitro Sensitivity of Torulopsis glabrata to Amphotericin B, 5-Fluorocytosine, and Clotrimazole (Bay 5097)

Thirty-five strains of Torulopsis glabrata were tested by a tube dilution method for their susceptibility to amphotericin B, 5-fluorocytosine, and clotrimazole (Bay 5097). Amphotericin B was the most active in vitro, inhibiting all strains at a concentration of 1 μg/ml and killing all strains at 2 μg/ml. 5-Fluorocytosine inhibited over 80% of strains at 0.24 μg/ml, but three strains required ≥7.8 μg/ml for killing. A concentration of 2 μg of clotrimazole per ml inhibited less than 50% of strains, and 8 μg/ml killed only 10% of strains. Most strains of T. glabrata were killed by therapeutically achievable concentrations of amphotericin B and 5-fluorocytosine, but not clotrimazole.