Band 65, Heft 1-2

Bioreduction of several prochiral carbonylic compounds such as acetophenone (1), ethyl acetoacetate (2) and ethyl phenylpropionate (3) to the corresponding optically active secalcohols 1a - 3a was performed using wild-type strains of Pichia pastoris UBB 1500, Rhodotorula sp., and Saccharomyces cerevisiae. The reductions showed moderate to excellent conversion and high enantiomeric excess, in an extremely mild and environmentally benign manner in aqueous medium, using glucose as cofactor regeneration system. The obtained alcohols follow Prelog’s rule, but in the reduction of 1 with P. pastoris UBB 1500 the anti- Prelog enantiopreference was observed

The mutagenicities of 2,2’-(di-3-hydroxyphenyl)-1H,1H’-[5,5’]-bisbenzimidazole, 2,2’-(di- 4-hy droxyphenyl)-1H,1H’-[5,5’]-bisbenzimidazole, 2,2’-(di-3-methoxyphenyl)-1H,1H’-[5,5’]- bisbenzimidazole, 2,2’-bis-(4-nitrophenyl)-1H,1H’-[5,5’]-bisbenzimidazole, 2,2’-bis-(3-nitrophenyl)- 1H,1H’-[5,5’]-bisbenzimidazole, 2,2’-bis-(4-methylphenyl)-1H,1H’-[5,5’]-bisbenzimidazole, 2,2’-(di-4-methoxyphenyl)-1H,1H’-[5,5’]-bisbenzimidazole, and 2,2’-bis-(3-me thylphenyl)- 1H,1H’-[5,5’]-bisbenzimidazole were studied in vitro using two strains of Salmonella typhimurium with frameshift mutation (TA98) and base-pair substitution mutation (TA100) as the plate incorporation assay in the absence of metabolic activation. These compounds are currently used to treat cancer. 4-Nitrophenyl and 3-nitrophenyl compounds were found to be mutagenic on both strains of Salmonella. A clear mutagenic response was seen in nitro-bound derivatives. The mutagenic response in Salmonella test strains (TA98, TA100) and structures of molecules suggest that nitro-bound molecules could be mutagenic.

A number of new substituted 1,2,4-triazole {[(1,2,4-triazolyl)ethyl]tetrazolyl} derivatives, their sugar hydrazones, and their acyclic C-nucleoside analogues were synthesized and tested for their antimicrobial activity against Bacillus subtilis (Gram-positive), Pseudomonas aeruginosa (Gram-negative), and Streptomyces species (Actinomycetes). The synthesized compounds displayed different degrees of antimicrobial activities or inhibitory actions.

A number of new substituted 1,2,4-triazole, 1,2,4-triazolo[3,4-b]1,3,4-thiadiazole and 1,2,4-triazolo[3,4-b]1,3,4-thiadiazine derivatives were synthesized and tested for their antimicrobial activity against Bacillus subtilis (Gram-positive), Pseudomonas aeruginosa (Gramnegative), and Streptomyces species (Actinomycetes). The synthesized compounds displayed different degrees of antimicrobial activities or inhibitory actions.

New acyclovir esters with peptidomimetics were synthesized and evaluated in vitro for their antiviral activity against the replication of Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The infl uence of peptidomimetics containing oxazole and thiazolyl-thiazole moieties on the antiviral activity is also reported. The esters were synthesized using the coupling reagents N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N,N-dimethyl-4-aminopyridine (DMAP) as a catalyst.

The development of new antibacterial compounds is an urgent issue to meet the evolution of resistivity of pathogenic bacteria against the available drugs. The objective of this study was to investigate the antibacterial compounds from the Antarctic lichen species Ramalina terebrata. A total of fi ve compounds, usnic acid, usimine A, usimine B, usimine C, and ramalin, were isolated by bioactivity guided-fractionation of the methanol extract of R. terebrata after several chromatographic procedures. The qualitative antibacterial activities of the crude extract and isolated compounds were determined by the disk diffusion method while the minimum inhibitory concentration (MIC) determination assay gave the quantitative strength of the test samples. All the test samples showed antibacterial activity against Bacillus subtilis. The crude extract and usnic acid showed antibacterial activity against Staphylococcus aureus. The MIC values of the isolated compounds against B. subtilis were in the range of 1 to 26 μg/mL. These observed experimental data showed the strong antibacterial potential of these compounds against B. subtilis.

A new ent-halimane-type diterpene, named 5(10),14-ent-halimadien-3β,13S-diol (1), was isolated from the bark of Amoora ouangliensis and its chemical structure determined on the basis of spectroscopic analysis. Additionally, ten other diterpenoids were obtained from A. ouangliensis and A. stellato-squamosa. The bioactive experiments of all compounds against AGZY 83-a (human lung cancer cells) and SMMC-7721 (human liver cancer cells) cells were documented.

Lichen phenolic compounds exhibit antioxidant, antimicrobial, antiproliferative, and cytotoxic activities. The purpose of this study was to evaluate the anticancer activity of lecanoric acid, a secondary metabolite of the lichen Parmotrema tinctorum, and its derivatives, orsellinates, obtained by structural modifi cation. A cytotoxicity assay was carried out in vitro with sulforhodamine B (SRB) using HEp-2 larynx carcinoma, MCF7 breast carcinoma, 786-0 kidney carcinoma, and B16-F10 murine melanoma cell lines, in addition to a normal (Vero) cell line in order to calculate the selectivity index of the compounds. n-Butyl orsellinate was the most active compound, with IC 50 values (the concentration that inhibits 50% of growth) ranging from 7.2 to 14.0 μg/mL, against all the cell lines tested. The compound was more active (IC 50 = 11.4 μg/mL) against B16-F10 cells than was cisplatin (12.5 μg/mL). Conversely, lecanoric acid and methyl orsellinate were less active against all cell lines, having an IC 50 value higher than 50 μg/mL. Ethyl orsellinate was more active against HEp-2 than against MCF7, 786-0, or B16-F10 cells. The same pattern was observed for n-propyl and n-butyl orsellinates. n-Pentyl orsellinate was less active than n-propyl or n-butyl orsellinates against HEp-2 cells. The orsellinate activity increased with chain elongation (from methyl to n-butyl), a likely consequence of an increase in lipophilicity. The results revealed that the structural modifi cation of lecanoric acid increases the cytotoxic activity of the derivatives tested.

A recent study showed that N-acylserotonin derivatives have strong inhibitory activity against tyrosinase. To clarify the role of the 5-hydroxy group in the indole ring, 2-, 4-, 5-, 6-, and 7-hydroxyindole and 11 related compounds such as 5-hydroxyindan and 6-hydroxyquinoline were tested for their inhibition of catecholase activity of tyrosinase from human HMVII melanoma cells. 6-Hydroxyindole (5) and 7-hydroxyindole (6) were potent inhibitors, while 5-hydroxyindole (4) was a weaker inhibitor than the above-mentioned compounds (IC 50 = 20, 79, 366, and 342 μM for 5, 6, 4, and kojic acid, respectively). 2-Hydroxycarbazole was also active (IC 50 = 190 μM), 5-hydroxyindan, 4-aminophenol, and harmalol were slightly active, and other compounds were inactive as an inhibitor. A similar pattern of inhibition was found with these compounds against mouse B16 melanoma tyrosinase, but with some differences from that for HMV-II tyrosinase. Kinetic analysis with HMV-II tyrosinase showed that the inhibition by hydroxyindoles 4, 5, and 6 was competitive with respect to the substrate LDOPA. Melanin formation in HMV-II cells was suppressed by 14% with 10 μM 5 without cytotoxicity, but 30 or 100 μM 5 decreased the cell viability. The present results suggest that 6-hydroxyindole is a potential and useful pharmacophore of antimelanogenic agents and that the position of a phenolic hydroxy group in a specifi c heterocyclic ring such as in indole is possibly optimized to yield more active inhibitors for tyrosinase.

Antioxidant properties of substrates [fl avanone (1), 6-hydroxy- (2), 7-hydroxy- (3), 5,7,4’- trihydroxy- (5), and 7-methoxyfl avanone (4)] and products of their microbial transformations, comprising hydroxylation, O-methylation, stereospecifi c reduction, dehydrogenation, and C-ring cleavage of the benzo-γ-pyrone system, were determined. Measurements of the antiradical activity (expressed as IC 50 value) of both the substrates and the products led to the determination of the impact of type and location of substituents in the tested fl avonoids on changes in their antioxidant activities.

Our objective was to investigate the concomitant production of protease and lipase by a bacterial strain. A promising bacterial strain was isolated from a food-processing industrial effl uent, which can produce both protease and lipase. The isolate was characterized by sequencing the16S rRNA gene. The PCR amplifi ed gene was subjected to analysis by BLAST to ascertain the genetic relatedness of the isolate, Bacillus pumilus SG2. The enzymes were produced and subjected to purifi cation by ammonium sulfate precipitation and dialysis followed by gel fi ltration chromatography; twelve-fold purity was obtained. The lipase produced was found to be proteolysis-resistant. The partially purifi ed enzymes were characterized for their optimum pH value, temperature, response to inhibitors, surfactants and oxidants. The relative molecular weights of protease and lipase were determined as 28 kDa and 40 kDa, respectively, by zymogram studies.

The indiscriminate use of wireless technologies, particularly of cell phones, has increased the health risks among living organisms including plants. We investigated the impact of cell phone electromagentic fi eld (EMF) radiations (power density, 8.55 μW cm-2) on germination, early growth, proteins and carbohydrate contents, and activities of some enzymes in Vigna radiata. Cell phone EMF radiations signifi cantly reduced the seedling length and dry weight of V. radiata after exposure for 0.5, 1, 2, and 4 h. Furthermore, the contents of proteins and carbohydrates were reduced in EMF-exposed plants. However, the activities of proteases, α-amylases, β-amylases, polyphenol oxidases, and peroxidases were enhanced in EMF-exposed radicles indicating their role in providing protection against EMF-induced stress. The study concludes that cell phone EMFs impair early growth of V. radiata seedlings by inducing biochemical changes.

Dark green islands (DGIs) are a common symptom of plants systemically infected with the mosaic virus. DGIs are clusters of green leaf cells that are free of virus but surrounded by yellow leaf tissue that is full of virus particles. In Cucumber mosaic virus (CMV)-infected Nicotiana tabacum leaves, the respiration and photosynthesis capabilities of DGIs and yellow leaf tissues were measured. The results showed that the cyanide-resistant respiration was enhanced in yellow leaf tissue and the photosynthesis was declined, while in DGIs they were less affected. The activities of the oxygen-scavenging enzymes catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in infected leaves were signifi cantly higher than those in the healthy leaves, and the enzyme activities in DGIs were always lower than in the yellow leaf tissues. Reactive oxygen species (ROS) staining showed that the hydrogen peroxide content in yellow leaf tissues was apparently higher than that in DGIs, while the superoxide content was on the contrary. Formation of DGIs may be a strategy of the host plants resistance to the CMV infection.

Clusia minor L., a C 3 -CAM intermediate, and Clusia multifl ora H. B. K., a C 3 obligate, present two physiotypes of a similar morphotype occurring sympatrically in the fi eld. Both species, exposed 2 days to high light, show similar responses to this kind of stress: (i) the level of xanthophyll pigments in tested plants during the daycourse adapts to stress, (ii) the levels of antheraxanthin and zeaxanthin clearly increase during the afternoon showing increased de-epoxidation, (iii) the changes in the xanthophyll cycle are similar. Exposure to high light increases the malate levels in C. minor during the afternoon while decreases the day/night changes of the malate levels, and hence the Crassulacean Acid Metabolism (CAM) expression. It can be concluded that strong light applied as a single stress factor to well-watered plants is not effective in strengthing the CAM metabolism in a C 3 -CAM intermediate plant but rather suppresses the CAM activity despite exposure to high light energy. It is suggested that, when water supply is not limiting and other stresses do not prevail, C 3 allows to use up the citrate pool, especially in the afternoon and enables a superior daily photon utilization.

The effects of Cd stress (200 μmol/L, 8 days) on respiration and photosynthesis of three wheat cultivars were investigated: Chuanyu 12 (CY12), Chuanmai 42 (CM42), and Chuanmai 47 (CM47). Fifteen-day-old seedlings were exposed to 200 μmol/L CdCl 2 for 4 days and 8 days, respectively. The results indicated that Cd was accumulated largely in roots, but little in leaves of all three cultivars. CY12 accumulated the highest level of Cd in roots and showed the weakest resistance. On the contrary, the other two cultivars, CM42 and CM47, adapted better to Cd stress, and their thiobarbituric acid-reactive substances (TBARS) contents were lower than in CY12, but the chlorophyll contents and water contents were higher than in CY12. Additionally, Cd stress prompted the alternative oxidase (AOX) activity and upregulated the cyanide-resistant respiration in CM42 and CM47 after 8 days; no such induction was observed for CY12. The CO 2 assimilation rate, leaf stomatal conductance and chlorophyll fl uorescence were inhibited by Cd stress in all cultivars, but more severe in the CY12 cultivar. Western blots indicated that the content of the photosystem II proteins LHCII and D1 decreased in CY12, but did not change in CM42 and CM47. While the content of the mitochondrial AOX protein increased markedly in CM42 and CM47, it did not in CY12. These results suggested that AOX and LHCII could be regarded as indicators of plant’s resistance to heavy metals.

Electrical impedance measurement - complex resistance in the presence of alternating current - is a useful tool for the investigation of structural characteristics of solid materials but also for plant tissues. This measurement is easily done: only two electrodes are inserted into the plant tissue, so it can be considered as a non-invasive technique and it may be a successful method for detecting structural changes in plants caused by environmental stresses. The effects of fl ood and cadmium stress were investigated by electrical impedance measurement, because both of them cause structural changes in plant tissues. Apoplasmic resistance (Ra), symplasmic resistance (Rs), and membrane capacitance (Cm) of pea roots were calculated. In the fi rst fi ve days of fl ood treatment, the Rs and Cm values of roots decreased drastically. In case of cadmium treatment, the Rs and Cm values of roots showed an increasing tendency supposedly as a consequence of the enhanced membrane rigidity, the thickened cell walls and decreased growth phenomena caused by the heavy metal. There also was a remarkable difference in cadmium accumulation patterns and in the changes of the calculated parameters amongst anoxic and aerated seedlings. This initial work revealed that the development of stress caused by two environental stress agents, cadmium and fl ood, can be followed by electrical impedance measurement.

A full-length cDNA of the carboxyltransferase (accA) gene of acetyl-coenzym A (acetyl- CoA) carboxylase from Jatropha curcas was cloned and sequenced. The gene with an open reading frame (ORF) of 1149 bp encodes a polypeptide of 383 amino acids, with a molecular mass of 41.9 kDa. Utilizing fl uorogenic real-time polymerase chain reaction (RT-PCR), the expression levels of the accA gene in leaves and fruits at early, middle and late stages under pH 7.0/8.0 and light/darkness stress were investigated. The expression levels of the accA gene in leaves at early, middle and late stages increased signifi cantly under pH 8.0 stress compared to pH 7.0. Similarly, the expression levels in fruits showed a signifi cant increase under darkness condition compared to the control. Under light stress, the expression levels in the fruits at early, middle and late stages showed the largest fl uctuations compared to those of the control. These fi ndings suggested that the expression levels of the accA gene are closely related to the growth conditions and developmental stages in the leaves and fruits of Jatropha curcas.

A reverse transcription-polymerase chain reaction (RT-PCR) strategy was used to clone diverse trypsin-like protease gene transcripts from midguts of the brown planthopper Nilaparvata lugens Stål (Homoptera: Delphacidae). Six individual trypsin-like protease transcripts were identifi ed. On the basis of one nucleotide sequence of the six clones, a full-length cDNA sequence (1902 bp) was obtained by rapid amplifi cation of cDNA ends (RACE). The cDNA contained an 1128-bp open reading frame encoding a putative protein of 375 amino acids with typical features of the trypsin-like protease. Heterogeneous expression of the coding sequence for the mature peptide in Escherichia coli cells showed that the expressed protease with a molecular weight of 27.0 is active, for its BApNAse activity assayed by using BApNA (N-benzoyl-D,L-arginine-p-nitroanilide) as substrate. The protease had its maximum activity at pH 8.0 and 35 °C. A much better stability was observed at pH values above 4.0 and temperatures below 40 °C. The enzyme was strongly inhibited by serine protease inhibitor. The trypsin-like protease is therefore likely one of the major digestive proteases responsible for protein hydrolysis in N. lugens gut, and multiple gene families encoding digestive proteases may help in adaptation of this sap-sucker to different rice varieties.

V-ATPase plays a central role in lepidopteran midgut ion transport physiology, and lepidopteran midgut turned out to be a model tissue for the study of V-ATPase. In the present study, the 5’-RACE method is used to obtain the 5’-UTR of V-ATPase c subunit gene from Bombyx mori. Sequence analysis of the promoter region and 3’-UTR of V-ATPase c subunit gene revealed that the transcription of the V-ATPase c subunit gene may be regulated by multi-ways. RT-PCR analysis showed that B. mori V-ATPase c subunit mRNA expresses in the whole developmental stages of B. mori. We also constructed a transient vector to determine the subcellular localization of the B. mori V-ATPase c subunit, and the result demonstrated that it is located in the membrane and some specifi c regions of BmN cells. Real-time PCR analysis further indicated that the c subunit mRNA expression was upregulated signifi cantly at 24 and 72 h in the midguts of resistant B. mori larvae after being inoculated with B. mori nucleopolyhedrovirus, suggesting that it may be related to the immune response against virus infection.

The solid-phase microextraction (SPME) technique was applied and optimized for collection of volatile compounds emitted from oestrous urine of mares Equs cabalus L. (Perissodactyla, Equidae) for GC-MS analyses. Variables such as type of SPME fi bre, collection time of volatiles, and addition of salt were optimized to improve the sampling effi ciency in two aspects: extent and selectivity of absorption/adsorption of urine volatiles onto SPME fi bres. The data revealed that the number of volatiles and the total amount represented as quantitative peak areas of the compounds trapped on fi bres coated either with polydimethylsiloxane- divinylbenzene or with divinylbenzene-carboxen-polydimethylsiloxane were signifi cantly higher compared to those coated with polydimethylsiloxane, polyacrylate, and carbowax-divinylbenzene. The polydimethylsiloxane-divinylbenzene-type of fi bre coating was chosen for optimization of sampling time and effect of salt addition. Sampling periods lasted for 15, 30, 60, 120, and 240 min. The optimal collection time of volatiles from urine maintained at about 36 °C was 60 min, as the number of compounds detected with amounts suffi cient for quantifi cation did not differ signifi cantly from those trapped during longer collection periods. No signifi cant increase in total amount of volatiles trapped was registered after 120 min of sampling. Addition of 0.3 g NaCl to the 2-ml of samples shortened the collection period from 60 to 15 min during which almost all compounds were trapped. Addition of salt has a signifi cant effect at all sampling periods taking into consideration the total amounts of volatiles trapped. The total intensities increased about 8, 5, 3, 3, and 2 times at collection periods of 15, 30, 60, 120, and 240 min, respectively, when compare with the ones obtained from the urine samples with no salt addition. In oestrous mare’s urine, 139 ± 4 (average number ± standard deviation) volatile compounds suitable for quantitative analyses were detected compared to 45 compounds collected by the gas-tight syringe method.

Strain T1E, isolated and identifi ed as Brevibacillus thermoruber, and evolutionally distant from the known keratinolytic isolates, proved to have feather-degrading ability. During the 7-day fermentation period, T1E consumed 10 g/l native goose feathers as the sole source of carbon and energy at 50 o C under aerobic conditions. The isolate secreted a thermostable, keratinolytic protease, which exhibited activity optimally at pH 6.5, whilst it was inhibited at alkaline pH. The keratin cleavage and catabolism resulted in the accumulation of free aspartic acid and soluble peptides with maximum values of 31.6 and 720 mg/l, respectively. The majority of the fermentation end-products were found to be small oligopeptides with an average molecular mass of 2275 Da.

The superoxide radical (O 2 ·̅), hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO) are pleiotropic infl ammatory mediators which play an important role in infl ammatory joint diseases. They are overproduced during rheumatoid arthritis and its experimental model - adjuvantinduced arthritis in rodents - and may be detected both systemically and intra-articularly. Their secretion is up-regulated by proinfl ammatory cytokines such as IFN-γ, IL-12, IL-6 and TNF-α, and they are responsible for the destruction of joint tissue. In this work, the effect of superoxide dismutase (SOD) from a thermotolerant yeast strain, Kluyveromyces marxianus, on the production of proinfl ammatory cytokines, reactive oxygen and nitrogen species was studied. Mice received three intraperitoneal injections of yeast SOD at a dose of 10 mg/ kg body weight (30,000 U/kg) on consecutive days starting on the day after arthritic induction. On days 3, 8 and 14 post induction peritoneal macrophages were isolated and both spontaneous and stimulated production of reactive oxygen and nitrogen metabolites were measured. Early in arthritic development yeast SOD treatment did not infl uence the O2·̅ production, but on day 14 both spontaneous and PMA-induced secretion were dramatically reduced. Spontaneous H 2 O 2 release was inhibited on day 14, while PMA-stimulated production was decreased from the beginning of the arthritic development. Yeast SOD treatment effectively suppressed the spontaneous and recombinant mouse IFN-γ + LPS induced release of NO as well. Serum levels of proinfl ammatory cytokines, IL-12, IFN-γ, IL-6 and TNF-α, were also signifi cantly reduced. The obtained results show some of the mechanisms of action of SOD in reducing the severity of arthritic infl ammation. Besides direct inhibition of joint tissue destruction exogenous SOD substantially limits the existing positive feedback between secretion of reactive oxygen species and infl ammatory cytokine production.

Ataxia telangiectasia mutated (ATM) kinase is a central player in cellular response to DNA damage. Phosphorylation of the histone H2AX by ATM is required for the accumulation of repair proteins at the sites of double-strand breaks. Recently, it was reported that the histone acetyltransferase Tat interactive protein-60 (TIP60) is required to acetylate ATM prior to its activation. The RuvB-like proteins TIP48 and TIP49 are known to be necessary for the assembly and functional activity of the TIP60 acetyltransferase complex. In the present communication, we investigated the requirements of TIP48 and TIP49 for ATM activation by monitoring the cell cycle distribution and H2AX phosphorylation after irradiation of TIP48- and TIP49-depleted cells. We found that neither the cell cycle nor γ-H2AX were affected in TIP48- and TIP49-silenced cells, suggesting that the TIP60 chromatin modifi cation complex is not engaged in DNA damage signaling upstream of ATM.

Using silkworm Bombyx mori Bm5 cells, we established a stable cell line expressing the human granulocyte macrophage colony-stimulating factor (hGM-CSF), which gets its name from the Bm5-hGM-CSF cell in which the glycoprotein of the hGM-CSF is secreted in the cell culture supernatant (CCS). It was demonstrated that secreted hGM-CSF had in vivo biological activity and the white blood cell (WBC) value increased two times that of the control. We expect to produce useful human recombinant glycoproteins from silkworm cultured cells for a low price and a large quantity