Volume 56, Issue 9-10

After the discovery of ‘movement proteins’ as a peculiarity of plant viruses and with the help of novel methods for the detection and isolation of interacting host proteins new insights have been obtained to understand the mechanisms of virus movement in plant tissues. Rapid progress in studying the molecular mechanisms of systemic spread of plant infecting viruses revealed an interrelation between virus movement and macromolecular trafficking in plant tissues. This article summarizes current explorations on plant virus movement proteins (MPs) and introduces the state of the art in the identification and isolation of MP interacting host proteins.

The structures of the pyoverdins isolated from the Pseudomonas spp. 96-312 and 96-318 were elucidated by spectroscopic and degradation techniques. As observed before for Pseudomonas spp. producing pyoverdins with a C-terminal cyclopeptidic substructure, the two strains can recognize to some extent structurally different pyoverdins as long as they have also a similar cyclopeptidic C-terminus.

From seven different pathovars of Pseudomonas syringae representing various genetic subgroups, and one strain of Pseudomonas viridiflava the same pyoverdin siderophore (1) was isolated, probably identical with the pyoverdin whose amino acid composition (but not their sequence) had been reported before. 1 is the first pyoverdin where two of the ligands for Fe 3+ are β-hydroxy Asp units. Its remarkably high complexing constant for Fe 3+ at pH 5 as compared with other pyoverdins offers a definite advantage in plant infection. The structure elucidation of 1 will be described and the taxonomical implications regarding pyoverdins with different structures ascribed previously to P. syringae strains will be discussed.

A new iridoid glucoside, 8-O-acetylshanzhiside (1), was isolated from the aerial parts of Phlomis tuberosa, together with two known iridoid glucosides, shanzhiside methyl ester and lamalbide. The known phenylethanoid glycosides acteoside and forsythoside B were also obtained and characterized. The structure of 1 was determined by means of 1D - and 2DNMR spectroscopic evidence.

One new, 3-O-{[[-α-ʟ-rhamnopyranosyl-(1→2)]-[β-ᴅ-glucopyranosyl-(1→3)]-β-ᴅ-glucopyranosyl-(1→2)]-β-ᴅ-glucuronopyranosyl}-3β,22α,24-trihydroxyolean-12-ene and two known triterpenoid saponins, 3-O-{[α-ʟ-rhamnopyranosyl-(1→2)]-[β-ᴅ-glucopyranosyl-(1→2)]-β-ᴅ-glucuronopyranosyl}- 3β,22β,24-trihydroxyolean-12-ene (azukisaponin V ) and 3,16-di-O-β-ᴅ-glucopyranosyl- 3β,6α,16β-trihydroxycycloart-24-ene have been isolated from Astragalus trigonus. The structures were determined primarily by NMR spectroscopy. The assignment of NMR signals was performed by means of 1 H - 1 H COSY, NOESY, ROESY , TOCSY, HMQC and HMBC experiments.

A methanolic extract and two fractions (n-hexane and ethyl acetate) from Virola oleifera leaves and some compounds (one lignan and two flavonoids) were investigated to verify the analgesic activity by using the writhing test in mice. The crude methanolic extract showed a moderate analgesic effect (about 40% of inhibition in this test at 10 mg/kg), whereas n-hexane and ethyl acetate fractions caused inhibition of 51.3 ± 5.9% and 50.5 ± 6.3% , respectively. Oleiferin-C (1), a lignan isolated from the n-hexane fraction, showed an interesting analgesic potential in this model when compared to two standard drugs, paracetamol (4-acetamidophenol) and aspirin (acetylsalicylic acid). The ID 50 calculated for this compound was 17.25 μmol/kg, with confidence interval between 13.7 and 21.3 μmol/kg, being about 8 times more potent than the standard drugs. The mixture of two glycoside-flavonoids, identified as astilbin (2) and quercitrin (3), also exhibited good analgesic activity, causing 63% of reduction of abdominal constriction in mice. These results suggest beneficial effect of this plant to treat dolorous processes.

Five known sesquiterpenoids, solavetivone, lubimin, lubiminoic acid, aethione and lubiminol were isolated from the root exudates recovered from Solanum aethiopicum by a newly proposed method using charcoal. Quantitative analysis of the sesquiterpenoids in the root exudates of S. aethiopicum and S. melongena suggested that relatively large amounts of the sesquiterpenoids were exuded from the roots. Antifungal activity of the sesquiterpenoids against Fusarium oxysporum and Verticillium dahliae was also examined.

A compound of unusual structure was isolated from red beetroot (Beta vulgaris) peel extract and identified as 5,5′ ,6,6′-tetrahydroxy-3,3′-biindolyl based on the combination of NMR and MS studies.

Preparative-scale separation of substituted anthraquinones by multiple liquid-liquid partition was studied using isopropylmethyl ketone (IM K)/aqueous phosphate buffer (aq.) as the solvent system and the Hietala apparatus with 100 partition units as the partition equipment. The lower (aq.) phase was chosen as mobile, while the upper (IMK) phase remained stationary. Hence, the principle of stepwise pH-gradient elution could be utilized to separate the components in two complex mixtures of hydroxyanthraquinones and hydroxyanthraquinone carboxylic acids, isolated from the fungus Dermocybe sanguinea. In spite of the nonlinearity of the partition isotherm s for these anthraquinones, implying deviations from the Nernst partition law, remarkable separations were achieved for the components in each mixture. Every anthraquinone carboxylic acid showed markedly irregular partition behavior, appearing in the effluent as two more or less resolved concentration zones. Such splitting was attributed to the formation of relatively stable sandwich-dimers, which were in a slow equilibrium with the monomers in the more nonpolar organic phase. At lower pH-values, only the polar monomers were distributed into the mobile aqueous phase and moved forward, whereas the nonpolar sandwich-dimers remained almost entirely in the stationary organic phase and lagged behind. When the pH of the mobile aqueous phase was raised high enough, the firmly linked dimers were monomerized and emerged from the apparatus as a second concentration profile. Intermolecular hydrogen bonding and π-π interaction between the n-systems of two anthraquinone molecules in a parallel orientation were considered responsible for the nonlinear and markedly irregular partition behavior of the natural anthraquinones studied. The nonlinearity of the partition behavior of the hydroxyanthraquinones lacking the carboxyl group, appeared merely as excessive broadening of the experimental concentration profile, which impaired the resolution between the components only insignificantly. Thus, e.g. the main components, dermocybin and emodin, could both be obtained from Separation 1 in a purity of at least 99% .

The terpenoid constituents of Targionia lorbeeriana grown in vivo and in vitro were com pared. The analysis of the dichloromethane extract was performed by HPLC-UV and by HPLC-M S. The obtained results revealed that the sesquiterpene lactones isolated from the dichloromethane extract of the wild Targionia lorbeeriana were also produced by the liverwort in in vitro cultures, in the same relative amounts. The composition of essential oils was evaluated by GC and GC-MS. Both, the yield and diversity of the essential oil obtained from wild growing T. lorbeeriana gametophytes were higher than those growing in vitro. Although, a significant number of compounds produced in vivo were maintained in vitro, a considerable number of other ones were not detected. Instead, under in vitro conditions, some new compounds were found which do not accumulate under wild conditions.

Satureja wiedemanniana (Lallem.) Velen (Lamiaceae) is an endemic species of Turkey. Aerial parts of the plant collected from eleven different localities in Turkey were subjected to hydrodistillation to yield essential oils which were analysed by a GC/MS system. Carvacrol, thymol, p-cymene and γ-terpinene were found as the main constituents. Antibacterial evaluation of the oils was also carried out.

From the flowers of meadowsweet and inflorescence of hawthorn the whole set of phenolic acids and flavonoids was analysed by TLC . Phenolic compounds were determined both as free ones and those liberated by hydrolysis. Moreover, ethyl ether and ethyl acetate extracts obtained from the analysed plants before and after alkaline and enzymatic hydrolyses were evaporated under reduced pressure and residues were analysed for their antioxidative properties. The weakest antioxidative activity was observed with the remaining residue after evaporation of ethyl ether extract obtained from enzymatically (β-glucosidase) hydrolysed hawthorn inflorescence water extract. The strongest antioxidative activity was noticed with the remaining residues after evaporation of ethyl ether extracts obtained from non-hydrolysed and hydrolysed in alkaline conditions of meadowsweet flower water extracts. The residues from meadowsweet flowers exhibited stronger antioxidative properties than residues obtained from hawthorn inflorescence and can be recommended as margarine preservatives.

Wild-type Arabidopsis L. leaves exposed to low ultraviolet-B (U V B ) conditions contained predominantly kaempferol glycosides, with low levels of quercetin glycosides. The flavonoid level doubled on treatment with UVB and an increase in the ratio of quercetin: kaempferol was observed. These results suggest that flavonols protect Arabidopsis plants from UVB damage, and indicate that the flavonoid 3’-hydroxylase (F3’H) enzyme, which converts dihydrokaempferol to dihydroquercetin, may play a crucial role. The tt7 mutant lacks this gene and, after treatment with sub-ambient UVB, contained kaempferol glycosides exclusively, to a level of total flavonols similar to that in wild-type Arabidopsis. Total flavonols after enhanced UVB treatment were higher in tt7 than in similarly treated wild-type plants, and only kaempferol glycosides were detected. Despite this high level, tt7 plants were less tolerant of UVB radiation than wild-type plants. These observations suggests that kaempferol is a less effective photoprotectant than quercetin. The chalcone isomerase (CHI) mutant (tt5) surprisingly did not accumulate naringenin chalcone, and this suggests that the mutation may not be restricted to the CHI gene alone. The concentration of hydroxycinnamic acid derivatives did not change with UVB treatment in most varieties indicating that their role in UV photoprotection may be subordinate to that of the flavonoids.

Photoactivation of the water splitting enzyme was performed with 13 different synthetic manganese complexes and characterized by oxygen evolution yield, thermoluminescence and chlorophyll fluorescence induction kinetics. The efficiency of different compounds in photoactivation correlated with the rate of linear electron transport in the presence of these com pounds. The organic ligands, associated with the manganese ions, do not prevent the photoactivation of the water splitting complex (WOC). Photoactivation with different manganese complexes depended on the number of the Mn-ions in the complex, their valence state and the nature of their donor atoms. The most efficient restorations were achieved by using tetram eric complexes having a dimer+dimer structure, complexes containing Mn(II) ions, and having 4-6 oxygen and 0-2 nitrogen atoms as donor atoms. Further, the effectiveness of photoactivation depended largely on the structure of the complexes. Our data support the notion that WOC in intact thylakoids requires the cooperation and well determined arrangement of all four manganese ions, and argue against the hypothesis that two manganese ions are sufficient for water splitting. Photoactivation by some complexes led to anomalous flashoxygen patterns, which are explained by a modified/perturbed water splitting complex.

Field investigations with Scots pine trees (Pinus sylvestris L.) were performed in eastern Germany, where ambient SO 2 , NO x and O 3 concentrations differed significantly in 1992-99 at three sites, namely Neuglobsow (yearly mean SO 2 in 1992: 9 μg m -3 ), Taura (yearly mean SO 2 in 1992: 54 μg m -3 and Rösa (yearly mean SO 2 in 1992: 73 μg m -3 ). To investigate the effects of SO 2 , NO x and O 3 on antioxidants (superoxide dismutase, ascorbic acid, glutathione, glutathione reductase, α-tocopherol) and pigments including chlorophyll fluorescence as well as visible damage symptoms in the form of needle yellowing and tip necroses, needles of the 1st and 2nd age class from young and mature trees were collected at the sites every October. Eight years after the start of the field study in 1992, the ambient SO 2 concentrations had decreased significantly at Neuglobsow (yearly mean SO 2 in 1999: 4 μg m -3 ), Taura (yearly mean SO 2 in 1999: 5 μg m -3 ) and Rösa (yearly mean SO 2 in 1999: 5 μg m -3 ). NO x and O 3 differed less at the three sites and showed no temporal variations. Whole needle glutathione continuously decreased, although concentrations were higher in needles of the 1 st and 2nd age class from the polluted sites Taura and Rösa than the unpolluted site Neuglobsow. The activities of glutathione reductase exhibited the same site-related differences and temporal variations and were correlated with concentrations of oxidized glutathione (GSSG). In contrast, the activities of the enzyme superoxide dismutase and the concentrations of whole needle ascorbic acid remained unchanged over the period. Only at the end of the investigation period did the concentrations of oxidized ascorbic acid (dehydroascorbate) increase in six-month-old needles at the polluted sites Taura and Rösa. Despite the clear decreases in SO 2 , the visible symptoms of needle tip necroses remained unchanged, especially at the polluted sites Taura and Rösa, although the needles contained higher pigment concentrations than needles from the unpolluted sites. The results of measurements with antioxidants as biomarkers for SO 2 -mediated stress in pine needles show that the adult Scots pine trees at the polluted sites suffered from greater oxidative stress than the needles from the less polluted site.

The rice herbicide cafenstrole and its analogs inhibited the incorporation of [1- 14 C]-oleate and [2- 14 C]-malonate into very-long-chain fatty acids (VLCFAs), using Scenedesmus cells and leek microsomes from Allium porrum. Although the precise mode of interaction of cafenstrole at the molecular level is not completely clarified by the present study, it is concluded that cafenstrole acts as a specific inhibitor of the microsomal elongase enzyme involved in the biosynthesis of fatty acids with alkyl chains longer than C 18 . For a strong VLCFA biosynthesis inhibition an -SO 2 - linkage of the 1,2,4-triazole-1-carboxamides was required. Furthermore, N,N-dialkyl substitution of the carbamoyl nitrogen and electron-donating groups such as methyl at the benzene ring of 1,2,4-triazole-1-carboxamides produced a strong inhibition of VLCFA formation. A correlation was found between the phytotoxic effect against barnyardgrass (Echinochloa oryzicola) and impaired VLCFA formation.

The production of 2,3-butanediol by Klebsiella pneumoniae from sugar cane juice supplemented with different salts was studied. This microorganism is able to degrade sucrose present in sugar cane juice containing ammonium phosphate as the sole nutritional supplement. With a sugar cane juice-based medium containing ~180 g sucrose /1 and 8.0 g (NH 4 ) 2 HPO 4 , / 1, over 70 g 2,3-butanediol plus acetoin / 1 were formed. This result is comparable to that achieved with a sugar cane juice-based medium containing several nutrients, although the kinetic profiles of these runs presented significant differences. With the ammonium phosphate- enriched medium, cell growth was initially favoured by both the strong oxygen supply and the higher water activity due to the lower concentration of nutrients. After 14 h, the limitation in some nutrients led to the interruption of cell growth, and decreasing rates for product formation and substrate consumption were observed. During the stationary phase of this run, sucrose was preferentially converted to product, and the substrate was completely depleted after 35 h of the process. With the complete medium, the substrate was totally consumed after 36 h of run. In this case, the higher initial concentration of nutrients reduced the overall process rate but sustained the cell growth for 27 h. Conversion yields of 0.40 g product / g sucrose and productivities close to 2.0 g/ lxh were obtained under both conditions.

The present work investigates the effect of α-, β- and γ-cyclodextrins (CD), i.e., α-CD, β- CD and γ-CD, on the oxygen evolution activity, the protein content and the uv-vis spectroscopic characteristics of thylakoid membranes. To study the pH-dependence, the thylakoids were incubated with the cyclodextrins at 273 K for a period of 10 min in the pH range from 5.5 to 9.0. To study the temperature-dependence the membranes were incubated at 273 and 293 K at pH 6.5, that is, the pH which induces a maximal oxygen evolution in the thylakoid preparations. The major observations are: (i) a stimulation of oxygen evolution in thylakoids incubated with α- and β-CD either in acidic or alkaline conditions, (ii) a low inhibitory effect induced by γ-CD on oxygen evolution, (iii) a significant decrease of the stimulatory effect of α- and β-CD on oxygen evolution as the incubation temperature is raised from 273 to 293 K, (iv) the apparent inability of the cyclodextrins to change the protein contents of the thylakoids, and (v) a significant CD-induced red-shift from 681 to 683 nm observed in the absorption and second derivative spectra of the thylakoid membranes treated with β-CD. First, it was found that the temperature effect described here is in accord with the general trend of the chemical effect of various cyclodextrins, i.e., the increase of the CD efficiency with decreasing temperature. Secondly, the CD effect is related to the size of the inner cavity diameter of the cyclodextrin molecules. An important conclusion in this work is that the molecular targets of the cyclodextrins are not limited to the thylakoid lipids as was described previously [Rawyler A. and Siegenthaler P.A. (1996) Biochim. Biophys. Acta 1278, 89-97], but are located as well in other molecular species exposed at the stromal side of the thylakoid membrane. In particular, the CD-induced red-shift from 681 to 683 nm in the absorption and second derivative spectra of the thylakoid membranes indicates that the cyclodextrins targets might be either the exposed heme macrocycle in cytochrome b559, or the chlorophylls and pheophytins in the pigment-proteins of the photosystems I and II.

Penicillium brevicompactum, Copper Uptake, Kinetics The copper binding properties of Penicillium brevicompactum biomass were influenced by growth phase of mycelium and concentration of copper in reaction mixtures. The efficiency of copper uptake increased with growth time and was largest at the mid-logarithmic growth phase. The time course of copper uptake was biphasic. Double reciprocal plots of absorption velocity of copper vs. copper concentration gave straight lines at concentration between 0.5 to 4 mᴍ . The apparent affinity of copper to the biomass of the stationary growth phase was the same as that of logarithmic growth phase and K m values were about 1.4 mᴍ . Pretreatment of the mycelium with glucose increased the amount of metal uptake about five times in comparison with the controls.

Isopenicillin N synthase (IPNS) is one of the key enzymes in the penicillin and cephalosporin biosynthetic pathway which catalyses the conversion of δ-(ʟ-α-aminoadipyl)-ʟ-cysteinyl-ᴅ-valine to isopenicillin N. The IPNS from Penicillium chrysogenum 23X-80-269-37-2, a high penicillin V-producer, was found to possess an isoleucine residue instead of tyrosine at position 195. An attempt to increase the specific activity of IPNS from Cephalosporium acremonium and Streptomyces clavuligerus was undertaken by altering the corresponding tyrosine residue to an isoleucine at the corresponding location. Unfortunately, no apparent increase in specific activity was encountered when the purified mutant enzymes were analysed and thus, this amino acid difference is likely not responsible for high specific activity in IPNS.

In this comparative study, three different mutagenesis kits, namely the MutaGene phagemid in vitro mutagenesis kit (Bio-Rad), the Transformerä Site-Directed mutagenesis kit (Clontech) and the Quik-change site-directed mutagenesis kit (Stratagene) were used for the mutagenesis of IPNS genes. However, a large difference in mutation efficiencies among these kits was encountered. Furthermore, these kits employ different strategies with its own individual strengths and weaknesses. Thus, a comparison among these three kits to evaluate their usefulness and improvements on the strategy adopted by the Quik-change site-directed mutagenesis kit, which was the kit of choice for our work, are presented for the benefit of research work.

The differential display reverse transcriptional polymerase chain reaction (DD-RT-PCR) was used to hunt for cDNA fragments specifically expressed by taxol treatment of HeLa cells. Forty-eight cDNA clones were differentially displayed through the experiments. The cDNA fragments obtained were separately spotted onto glass slides to prepare a tailor-made DNA chip. The gene expression pattern of differentially displayed cDNA fragments were checked by DNA microarray analysis.

In this study we report the effects of sulfated polysaccharides on the ecto-ATPase activity of intact cells of Leishmania tropica. Increasing concentrations of dextran sulfate stimulated progressively the ecto-ATPase activity, but did not modify other ecto-enzymes present on the surface of this parasite, such as 5′ nucleotidase, 3′nucleotidase and a membrane-bound acid phosphatase activity. This stimulation was not observed when other sulfated polysaccharides such as chondroitin sulfates and heparin were tested. It depends on size and charge of the dextran sulfated molecule. When the cells were incubated in the presence of dextran sulfate Mr 8,000; 40,000 and 500,000 the stimulation of the ecto-ATPase activity was 11%; 23%; and 63%, respectively, and the stimulation was not observed when desulfated dextran (Mr 40,000) was used. The effects of dextran sulfate also depend on pH of the medium. At pH 7.5, the stimulation was over 60%, whereas at pH 8.5 only 25%. The effects of dextran sulfate 500,000 on the ecto-ATPase activity was totally abolished by spermidine and partially by putrescine, two polyamines synthesized and released by Leishmania.

Monolayer Technique, Nonionic Detergent, Erythrocyte Shape The degree and time-course of expansion of palmitoyloleoylphosphatidylcholine (PC) and bovine brain phosphatidylserine (PS)/PC (75:25, mol/mol) monolayers at 32 mN/m caused by differently charged amphiphiles (detergents) added to the sub-phase buffer (pH 7.4, 22 °C) were followed. Amphiphiles were added to the sub-phase at a concentration/monolayer area corresponding to the concentration/erythrocytes surface area where sphero-echinocytic or sphero-stomatocytic shapes are induced (0.46-14.6 μᴍ). Nonionic, cationic and anionic am­ phiphiles expanded the PS/PC monolayer significantly more (1.7-4.2 times) than the PC monolayer. A zwitterionic amphiphile expanded both monolayers to a similar extent. The initial rate of monolayer-expansion was higher for all amphiphiles (1.7-20.4 times) in the PS/PC monolayer than in the PC monolayer. It is suggested that hydrophobic interactions govern the intercalation of amphiphiles into monolayers, and that monolayer packing, modulated by phospholipid head group interactions and alkyl chain saturation, strongly influence amphiphile intercalation. A possible relation between the monolayer-expanding effect of amphiphiles and their effect on erythrocyte shape is discussed.

Biological effects of weak magnetic fields are widespread, but poorly understood. Besides magnetic particles, which have been shown to be involved in only few cases, membranes are discussed as the site of perception. However, the mechanism is unknown. We have subjected pure lipid membranes to weak magnetic pulses, and found, that their electric properties are modified. Black lipid membranes were prepared from purified asolectin on a teflon septum separating electrically the two chambers of a teflon cuvette, using the technique of Mueller et al. (1962). Single magnetic pulses were applied for 10 μs, whose intensity could be varied from 0 to 100 G (0 to 10 mT) at the membrane. Directly after the pulse decay, the conductance of the bilayers was scanned with 10 periods of a 1 kHz triangle alternating voltage (eg. a measurement time window of 10 ms). Frequency spectra of the bilayer current rose by a frequency dependent factor ≤ 2 in a broad region around 80 kHz, when the amplitude of the preceding magnetic pulse was increased from 0 to 100 G. The data show, that weak magnetic fields can significantly change the electrical conductance of lipid films. The relaxation of electrons in a two-dimensional quantum state (“quantum hollow”) will be discussed as a possible origin of these effects.

The effect of novel synthesized aminophosphonates on membrane potential and electrical conductance of internodal cells of Nitellopsis obtusa and hemolysis of erythrocytes (RBC) was studied. It was found that those the organophosphorous compounds, when present at 10-100 μm concentrations, caused depolarization and increased electrical conductance of alga membranes. They also influenced fluidity of erythrocyte membranes. When used at higher concentrations aminophosphonates caused hemolysis of RBC. The changes observed depended on structural features of the aminophosphonates, i.e., substituents at the carbon, phosphorus and nitrogen atoms, and, most probably, may be the result of direct interaction of the am inophosphonates with the lipid phase of the plasma membrane and the induced structural changes. Two modes of interaction are proposed.

The active site of Viviparus ater (mollusc) hemocyanin was investigated using the fact that the binding of dioxygen to the binuclear copper-containing sites of hemocyanins is connected with the appearance of specific dichroic bands which are very sensitive to changes in the structrure and polarity of the environment. Oxy-Viviparus ater hemocyanin exhibits near UV and visible circular dichroism spectra different from those of other molluscan and arthropo-dan hemocyanins. These differences are due probably to variations in the geometry or charge distribution in the dioxygen binding sites of the compared proteins. The thermostability of Viviparus ater hemocyanin and the significance of the copper-dioxy-gen system for the stability were also investigated. “Melting” temperatures, T m , of 77 °C for the oxy-hemocyanin and 57 °C for the apo-protein were calculated from the denaturation curves which demonstrates the considerable role of the binuclear active site for the thermostability. Viviparus ater hemocyanin is more thermostable than other hemocyanins for which data are published.

Metallothioneins levels (MTs) in the clams Callista chione and Venus verrucosa, collected from two coastal sites in Greece, were determined and quantified by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and a spectrophotometric assay (Ellm an’s reaction). SDS-AGE separation in the digestive gland, which represents the hepato-pancreas in clams, demonstrated the presence of MTs similar to mammalian MT (rabbit liver Cd, Zn-thionein). No other SH-containing proteins apart from the MTs were detected. MT levels quantified by the Ellman’s reaction indicated seasonal variation for both species. The highest values were recorded in the spring and the lowest in the autumn. The seasonal variation and the differences in the MT levels of the two areas seem to be related to the reproductive cycle of the organisms as well as to abiotic factors of each area. Our results show that both C. chione and V. verrucosa have the potential to be used as biomarkers of metal pollution, provided that the influence of the external factors is safely quantified.

The influence of some inorganic (K + , Mg 2+ , and Al 3+ ) and organic C n H 2n+1 SO 3 -, n=12 and 14) ions was studied on the hemolysis of erythrocytes (RBC) caused by organolead compounds (tripropyllead -TPL, tributyllead -TBL and triphenyllead -TPhL chlorides). It was found that sulfonate anions increased the hemolytic effect induced by triorganoleads, while inorganic cations protected RBC against the triorganoleads action, especially when the latter were used at small concentrations. This protection was weaker when the concentration of organoleads increased and depended on the kind of ion. The protective efficacy sequence was like that: Mg 2+ > Al 3+ > K + . The less hemolytic of the triorganoleads studied was TPL. TBL was slightly more effective than TPhL. The efficacy of the sulfonate ions to increase the triorganolead chloride -induced hemolysis was practically the same for TPL and TBL. A weaker efficacy of C 12 H 25 SO 3+ was observed when TPhL was used as RBC membrane modifier.

Alterations in the electrical passive parameters of red blood cell membranes occurring during storage have been investigated by means of two different experimental approaches, i.e., radiowave dielectric spectroscopy measurements and flow-cytometric measurements. We observed a correlation between the appearance of phosphatidylserine molecules in the outer leaflet of the cell membrane and the occurrence of a change in the electrical passive membrane parameters. The electrical re-organization of the membrane, resulting in an increase of its conductivity and permittivity after 5 -7 days from blood storage, can be considered as a precursory event for the loss of asymmetry in the lipid distribution across red blood cell membrane.

The electron densities of the atoms in Fe(II)-or Al(III)-tyrosine hydroxylase (THO) containing oxygen and pterin were calculated by the DFT (Density-functional theory) method. The results obtained are consistent with our previous proposal that oxygen activation in THO proceeds through the formation of an intermediate derived from Fe(II), oxygen, and pterin. Electron donation from substrate to the oxygen molecule is important to cleave the O-O bond, and to give the hydroxylated product. Based on these results, it was concluded that hydroxylation of the aromatic ring does not proceed in the Al(III)-containing THO, and a relationship exists between Al(III) ion and neurodegeneration.

Interaction between the plasma membrane and aggregate lipid surface determines how efficiently the encapsulated drug will be delivered into the cell. Electrostatic interactions are one of the main forces affecting liposome and aggregate association with the charged cell surface. In this study, the effect of surface charge on the association of liposomes with human colon CX-1.1 cancer cells was studied. When phosphatidylserine was incorporated into a lipid bilayer, the amount of liposomes associated with cells tended to increase along with the amount of negatively charged lipid present in the liposomal lipid bilayer. When the cationic lipid dioleoyl-1,2-diacyl-3-trimethylammonium-propane (DOTAP) was included into the liposome formula, their uptake by the cells was also increased. Maximum binding occurred when the amount of positively charged lipids in liposomes was about 10 mol% of lipids.

Three new groups of phenolic antioxidants, quaternary ammonium salts with a phenol ring and alkyl chains of different length (pyrrolidine ethyl esters of 3,5-di-t-butyl-4-hydroxydihydrocinnamic acid n-alkoxymethylchlorides (PYE-n) or n-alkylbromides (PYA-n) and 2-dimethylaminoethyl ester n-alkylbromides (PPA-n), were synthesized. Some of them were previously found to efficiently protect yeast cells against oxidants and to inhibit the production of thiobarbituric acid-reactive substances in whole yeast cells and in isolated membrane lipids. The new antioxidants (at 1-100 μm) abolished or diminished peroxidation of oliwe oil emulsions caused by the OH • -producing Fe 2+ and RO • and ROO • -producing tertbutylhydroperoxide (TBHP) and the azo compounds 2,2′-azobis-(amidinopropane)dihydronitrile (AAPH) and 1,1′-azobis-(1-cyclohexanecarbonitrile) (ACHN): all present at 10 mᴍ . The efficiency of individual both antioxidants was examined in relation to the type of lipid peroxidation inducer, the site of antioxidant incorporation into the emulsion lipid phase, the length of the alkyl chain, and the maximum concentration of effective antioxidant monomers given by its critical micelle concentration. PYA-n class compounds were highly efficient against all peroxidation inducers and their efficiency did not depend on the position of their molecules in the lipid phase and/or on the aliphatic chain length. In contrast, the efficiency of PYE-n and PPA-n class compounds depended both on the type of oxidant and on the length of their aliphatic chain. Their potency against Fe 2+ and ACHN increased with increasing alkyl chain length whereas with AAPH it dropped with increasing alkyl chain length. A similar pattern was found with the action of PYE-n against TBHP whereas in the PPA-n group an extending alkyl chain reduced the anti-TBHP efficiency. These relationships may not be entirely straightforward and other factors (chemical nature of each compound, its possible interaction with fluorescent probes used for diagnostics, etc.) may play a considerable and not yet quite clear role. PPA-n class antioxidants have the lowest critical micelle concentration, which may limit their efficiency. Nevertheless, these phenolic antioxidants can be conveniently employed as highly efficient inhibitors of lipid peroxidation.

The effects of DNA interacting drugs on: (1) total RNA synthesis catalyzed by E.coli and T7 RNA polymerase; (2) synthesis of the initiating dinucleotide (pppApU) by E .coli RNA polymerase (“abortive initiation“); (3) elongation of RNA chains synthesized by T7 RNA polymerase on pT7-7 plasmid DNA bearing T7 RNA polymerase promoter ϕ 10 with human Cu/Zn superoxide dismutase coding sequence, (4) interaction of transcription factor Sp1 and its binding site were studied. Intercalating ligands which form quickly dissociating complexes with DNA (anthracyclines, proflavine, ethidium bromide) are compared with the slowly dissociating drug of d(G · C ) specificity (actinomycin D), the non-intercalating, d(A · T ) specific pyrrole antibiotics (netropsin and distamycin A) and covalently binding to DNA 1-nitroacridine derivative (nitracrine). The obtained results indicate that rapidly dissociating ligands, proflavine and ethidium bromide, inhibit total RNA synthesis in vitro and the abortive initiation to a similar extent while they do not induce discrete elongation stops of RNA polymerase. Actinomycin D and nitracrine exhibit a high inhibitory effect on total RNA synthesis and induce stops of RNA polymerase while not affecting abortive initiation. Pyrrole antibiotics primarily inhibit the initiation, while no elongation stops are induced. Actinomycin D inhibits complex formation between nuclear proteins and the Sp1 binding site. Netropsin, ethidium bromide, proflavine and other intercalating acridines do not affect Sp1 binding. The results indicate that the effects primarily depend on sequence specificity and secondarily on the dissociation rate of ligands from their complexes with DNA.

The combination of cis-diamminedichloroplatinum (II) (DDP, cisplatin) and topoisomerase II inhibitor teniposide (VM-26) has been shown to exert a synergistic effect in the clinical treatment of cancer. In this study, the combined effect of DDP and VM-26 on the growth and induction of apoptosis in synchronized murine erythroleukemia (MEL) cells, treated at the beginning or in the middle of S-phase of cell cycle, was examined. MEL cells, clone F4 N, were synchronized by a double thymidine block leading to accumulation of 70% of cells at the G 1 /S boundary. The growth-inhibitory effect of DDP and VM-26 applied alone were stronger in the middle of the S-phase than at the beginning. Morphological analysis showed that the majority of the cells revealed typical signs of apoptosis: nuclei fragmentation and appearance of apoptotic bodies. The combination of both agents at low concentrations had a synergistic effect on cytotoxicity. At higher concentrations the effect was additive. The remainder of the cells were characterized by unbalanced growth, aberrant mitosis and appearance of multinucleated cells. These processes led to delayed cell death. The appearance of aberrant mitosis was more expressed after treatment in the middle of the S-phase. It is likely that as a result of the combined action of cisplatin and VM-26, cells become supersensitive to the ability of topoisomerase II inhibitor to influence mitosis, and this increased sensitivity may contribute to the observed synergism.

The susceptibility of the small abalone Haliotis diversicolor supertexta to Vibrio parahae­molyticus 880915 strain and its extracellular products (ECP) at different temperatures was investigated. The strain was previously isolated from the haemolymph of the moribund small abalone with withering syndrome during an outbreak of mass mortality among the cultured animals in September 1999 in I-Lan, Taiwan. The bacterium and its ECP were lethal to the small abalone. Onset of the withering syndrome in the moribund or dead animals could be observed at 4 -7 d post-bacterial challenge. The same bacterial strain could be isolated from the haemolymph of the moribund animals with or without the syndrome post-bacterial challenge. This syndrome could not be observed in the moribund or dead animals post-ECP challenge. The animals were more susceptible to the bacterium and ECP challenge at higher temperature (28 °C) indicating that the outbreak of the disease in warmer season is associated with thermal induction.

Monomelittoside, melittoside, 8-acetyl-harpagide, har­pagide, ajugol, 5-desoxy-harpagide, 5-desoxy-8-acetyl-harpagide and catalpol were isolated from the aerial parts of S. grandidentata.

A re-investigation of subaerial parts of Leontodon cichoraceus (Ten.) Sanguin. yielded the new germacrane- type sesquiterpenoid 15-β-D-glucopyranosyl-8- [p-(β-D-glucopyranosyloxy)phenylacetyl]-salonitenolide. The structure was established by APCI mass spectrometry and 1D- and 2D-NMR spectroscopy. A survey by HPLC-DAD and HPLC-MS gave no signs of this compound in 23 other taxa of Leontodon.

One of the perspective methods of cancer chemotherapy is magnetic targeting of drugs to tumors. This task is usually accomplished using small permanent magnets attached near the desired sites. In this study a new much more effective approach is proposed which is based on a strong magnetic gradient using a ferromagnetic wire placed in a strong magnetic field. Feasibility of this approach has been evaluated by the formation of ferrofluid seals and measurement of maximum pressure the formed seal can resists. Using this method it is possible to capture even superparamagnetic particles with nanos­ize dimensions, therefore the method may have an interesting applications in biomedical sciences.

The effects of different a-D-Glucose (Glu) concentrations (0 -16 mᴍ) on Na + , K + -ATPase and Mg 2+ -ATPase activities were investigated in homogenates of adult male rat whole brain at 37 °C. The enzyme activities were determined after 1h preincubation with Glu. Brain Na + , K + -ATPase was not affected by Glu different concentrations. On the contrary, Mg 2+ -ATPase activity was considerably reduced with Glu concentrations lower than 4 mᴍ. The enzyme was inhibited 40%, 50% or 80% with 3, 2 or 1 mᴍ of Glu, respectively. The above results suggest: a) The various concentrations of Glu have no effect on brain Na + , K + -ATPase activity, b) The inhibited brain Mg 2+ -ATPase in hypoglycemia produces low intracellular Mg 2+ , which could modulate the activity of Mg 2+ -dependent enzymes and the rates of protein syn­thesis and growth of the cell.

The function of gene sll0033 from Synechocystis 6803 which is homologous to the bacterial crtI-type phytoene desaturase genes was elucidated as a novel carotene isomerase. Escherichia coli transformed with all genes necessary for the formation of ζ-carotene and expressing a ζ-carotene desaturase synthesized the positional isomer prolycopene (7,9,7′,9′Z lycopene) which cannot be cyclized in the subsequent reactions to a- and β-carotene. Upon cotransformation with sll0033, the formation of all-E lycopene is mediated instead.

The aim of this study is to find out the ratio of Nosema infected honey bees which are producing the famous Anzer honey that is used for the cure of the illnesses such as farangitis, tonsilitis, ulceration, and scratchs due to the experiences of the people living in Turkey. Honey bee samples were collected from two different regions of Anzer plain in July. Honey bee abdomens were homogenized and 1 ml distilled water was added for each honey bee. Later, 0.1 ml out of this solution was examined by Neubauer slides and the number of Nosema apis spores were counted. The results showed that Nosema apis significantly infected the honey bees although it was summer season. However, the summer season at the Anzer plain, when compared with the Mediterranean climate, is considered to be spring.