New expanded bed adsorbents for the recovery of DNA

A 20–40 m pellicular high density (3.7 g cm^–3) expanded bed material has been designed for the capture of DNA and other large macromolecules. Anion exchangers fashioned out of these supports exhibited dramatically enhanced DNA binding capacities over commercial anion exchange adsorbents (6 mg ml^–1, c.f. 50 g ml^–1 at 10% breakthrough), due to a combination of small particle and fuzzy surface architecture created through the coupling of polyethylene imine chains.     

Genetical studies on the heterocyst and nitrogen fixation of the blue-green alga Nostoc muscorum

Summary The short-trichome-forming, non-heterocystous and non-nitrogen-fixing (het ^– nif^–) mutant of the nitrogen-fixing blue-green alga Nostoc muscorum was isolated by N-methyl-N-nitro-N-nitrosoguanidine (NTG)-mutagenesis after penicillin enrichment technique and characterized. The mutant did not grow and fix nitrogen in combined-nitrogen-free medium while in nitrate-containing medium it grew well (K=0.112/day, G=64.27 h), although its growth was comparatively poor than the parent alga (K=0.128/day; G=56.14 h). The mutant was stable and both the het ^– and nif ^– characters reverted to wild type (het ⁺ nif⁺) with the reversion frequency of 2.62×10^-7.The het ^– nif^– mutant tolerated 0.5 g/ml of streptomycin sulphate on the agar medium and its streptomycin resistant mutant capable of growing in presence of 10g/ml of streptomycin was isolated spontaneously with a frequency of 1.45×10^-8. These streptomycin resistant isolates (het ^– nif^– str^R) resisted 100 g/ml of streptomycin sulphate on the agar medium and 200 g/ml in liquid medium. Spontaneous virus-resistant mutant of het ^– nif^– str^R was isolated with a mutation frequency of 4.02×10^-4.The data of genetic recombination experiments suggested that there is transfer of both het and nif genes to het ^– nif^– strain with the frequency of 2×10^-6 to 2×10^-5 simultaneously. There was increase in recombination frequency with increasing the incubation period. The virus-resistance marker is also transferred to the sensitive recipient.Abbreviations CFU colony forming units - C–N Chu-10 medium without combined nitrogen - C+N Chu-10 medium with 0.232 g/l calcium nitrate - G generation time - het heterocyst differentiating genes - K specific growth rate constant - MOI multiplicity of infection - nif nitrogen-fixing genes - NTG N-methyl-N-nitro-N-nitrosoguanidine - PFU plaque forming units - str ^R streptomycin resistance - str ^R streptomycin sensitive     

First survey on the natural occurrence of Fusarium mycotoxins in Bulgarian wheat

Wheat for human consumption (140 samples) was collected after harvest from all regions of Bulgaria. The 1995 crop year was characterized by heavy rainfall in the spring and summer months. The internal mycoflora of wheat samples was dominated by Fusarium spp. and Alternaria spp., and storage fungi were rarely present. The samples were analysed for contamination with Fusarium mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), and zearalenone (ZEA), using enzyme immunoassay methods. DON and ZEA were the predominant toxins, with a contamination frequency of 67% and 69%, respectively. The average levels of these toxins in positive samples were 180 g/kg (DON) and 17 g/kg (ZEA), maximum concentrations were 1800 g kg^–1 and 120 g kg^–1, respectively. Acetyl derivatives of DON, namely 3-AcDON and 15-AcDON, were found in 2.1 % and 0.7% of the samples, at at maximum level of about 100 g kg^–1. Only one sample was positive for T-2 (55 g/kg), DAS was not detected. This is the first report about the natural occurrence of a range of Fusarium mycotoxins in wheat for human consumption in Bulgaria.Abbreviations 3-AcDON 3-acetyldeoxynivalenol - 15-AcDON 15-acetyldeoxynivalenol - DAS diacetoxyscirpenol - DON deoxynivalenol - EIA enzyme immunoassay - T-2 T-2 toxin - ZEA zearalenone     

In vitro analysis of ion channels in periaxolemmal-myelin and white matter clathrin coated vesicles: Modulation by calcium and GTPγS

This study reports the analysis of K⁺ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K⁺ channels were observed with amplitudes of 25–30, 45–55, and 80–100 pS, all of which exhibited mean open-times of 1–2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K⁺ channel activity was regulated by Ca²⁺. Little or no change in activity was observed when Ca²⁺ was added to thecis side of the bilayer. Addition of 10 M total Ca²⁺ also resulted in little change in K⁺ channel activity. However, at 80 M total Ca²⁺ all K⁺ channel activity was suppressed along with the activation of a 100 pS Cl^– channel. The K⁺ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTPS to thetrans side of the bilayer resulted in a restriction of activity to the 45–50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K⁺ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the subunits of G₀, G_s, and G_i and the low molecular weight G protein,ras. As with periaxolemmal-myelin treated with GTPS, the vesicle membrane exhibited only the 50 pS channel. The channel was active at all holding potentials and had open times of 1–6 msec. Addition of GTPS to the bilayer fused with vesicle membrane appeared to suppress this channel activity at low voltages yet induced a hyperactive state at holding potentials of 45 mV or greater. The vesicle 50 pS K⁺ channel was also activated by the 6-methyl-dihydropyron-2-one (20 M).Abbreviations CNPase 2–3 cyclic nucleotide phosphohydrolase - EDTA ethylenediamine N,N,N,N-tetraacetic acid - G-protein GTP(guanosine triphosphate) binding protein - GTPS guanosine 5-O-(3-thiotriphosphate) - MAG myelin associated glycoprotein - Na⁺ K⁺ ATPase, Na⁺ and K⁺ stimulated adenosine triphosphatase - PLP myelin proteolipid protein Special issue dedicated to Dr. Majorie B. Lees.     

Interrelationships Between Nitrogen Supply and Photosynthetic Parameters in Vicia faba L.

We determined for Vicia faba L the influence of nitrogen uptake and accumulation on the values of photon saturated net photosynthetic rate (P _Nmax), quantum yield efficiency (), intercellular CO₂ concentration (C _i), and carboxylation efficiency (C _e). As leaf nitrogen content (N_L) increased, the converged onto a maximum asymptotic value of 0.0664±0.0049 mol(CO₂) mol(quantum)^–1. Also, as N_L increased the C _i value fell to an asymptotic minimum of 115.80±1.59 mol mol^–1, and C _e converged onto a maximum asymptotic value of 1.645±0.054 mol(CO₂) m^–2 s^–1 Pa^–1 and declined to zero at a N_L-intercept equal to 0.596±0.096 g(N) m^–2. fell to zero for an N_L-intercept of 0.660±0.052 g(N) m^–2. As N_L increased, the value of P _Nmax converged onto a maximum asymptotic value of 33.400±2.563 mol(CO₂) m^–2 s^–1. P _N fell to zero for an N_L-intercept of 0.710±0.035 g(N) m^–2. Under variable daily meteorological conditions the values for N_L, specific leaf area (_L), root mass fraction (R_f), P _Nmax, and remained constant for a given N supply. A monotonic decline in the steady-state value of R_f occurred with increasing N supply. _L increased with increasing N supply or with increasing N_L.     

Solution structure of a synthetic peptide corresponding to a receptor binding region of FSH (hFSH-beta 33-53).

The receptor binding surface of human follicle-stimulating hormone (hFSH) is mimicked by synthetic peptides corresponding to the hFSH- chain amino acid sequences 33–53 [Santa-Coloma, T. A., Dattatreyamurty, D., and Reichert, L. E., Jr. (1990),Biochemistry 29, 1194–1200], 81–95 [Santa-Coloma, T. A., and Reichert, L. E., Jr. (1990),J. Biol. Chem. 265, 5037–5042], and the combined sequence (33–53)–(81–95) [Santa-Coloma, T. A., Crabb, J. W., and Reichert, L. E., Jr. (1991),Mol. Cell. Endocrinol. 78, 197–204]. These peptides have been shown to inhibit binding of hFSH to its receptor. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy were used to determine the structure of the first peptide in this series, the 21 amino acid peptide hFSH--(33–53), H₂N-YTRDLVYKDPARPKIQKTCTF-COOH. Analysis of CD data indicated the presence of approximately equal amounts of antiparallel -pleated sheet, turns including a -turn, other structures, and a small amount ofa-helix. The major characteristics of the structure were found to be relatively stable at acidicpH and the predominant effect of increased solvent polarity was a small increase ina-helical content. One- and two-dimensional NMR techniques were used to obtain full proton and carbon signal assignments in aqueous solution atpH 3.1. Analysis of NMR results confirmed the presence of the structural features revealed by CD analysis and provided a detailed picture of the secondary structural elements and global folding pattern in hFSH--(33–53). These features included an antiparallel -sheet (residues 38–51 and 46–48), turns within residues 41–46, and 50–52 (a -turn) and a small N-terminal helical region comprised of amino acids 34–36. One of the turns is facilitated by prolines 42 and 45. Proline-45 was constrained to thetrans conformation, whereas proline-42 favored thetrans conformer (70%) over thecis (30%). Two resonances were observed for the single alanine residue (A-43) sequentially proximal to P-42, but the rest of the structure was minimally affected by the isomerization at proline-42. The major population of molecules, containingtrans-42 andtrans-45 prolines, presented 120 NOEs. Distance geometry calculations with 140 distance constraints and energy minimization refinements were used to derive a moderately well-defined model of the peptide's structure. The hFSH--(33–53) structure has a highly polar surface composed of six cationic amino acid (arginie-35, lysine-40, arginine-44, lysine-46, glutamine-48, and lysine-49) and two anionic residues (aspartate-36 and aspartic acid-41). A hydrophobic region in the structure is composed of residues in the antiparallel -sheet and -turn which fold to produce a distorted hairpin. The structure of this domain, together with the protruding and positively charged region in the vicinity of residues 42–45, may mimic the surface of hFSH that binds to the receptor.Abreviations used: hFSH, human follicle-stimulating hormone; PB, 25 mM Na₂KPO₄, 25 mM KH₂PO₄, and 5 mM Mg Cl₂; CD, circular dichroism spectrapolarimetry; NMR, nuclear magnetic resonance spectrometry; COSY, homonuclear correlated spectroscopy; NOESY, 2D nuclear Overhauser effect spectroscopy; HOHAHA, homonuclear Hartman-Han coherence transfer; HMQCHY, reverse-detected heteronuclear multiple shift correlation, one bond; HMBC, reverse-detected heteronuclear multiple bond correlation; S/N, signal to noise ratio; TFE, trifluoroethanol.Dr. Santa-Coloma is on leave of absence from the National Research Council of Argentina (CONICET).     

Feeding rate inhibition in crowded Daphnia pulex

Feeding rates of Daphnia pulex fed a range of levels of the alga Chlamydomonas reinhardi of 15 °C are strongly density-dependent. At lower densities, Daphnia (30 1^–1) fed at higher rates than crowded (270 1^–1) Daphnia which manifest a relatively depressed saturation feeding response. At 30 individuals/liter, Daphnia consumed 8.5 – 15.7 × 10⁴ cells d^–1h^–1 (on a volume basis, 12.1 – 22.2 × 10⁶ m³), at 270 L^–1 3.7 – 3.9 × 10⁴ (5.2 – 5.5 = 10⁶ m³ cells d^–1h^–1 when feeding on algae at 80 000 cells ml^–1 (11.3 × 10⁶ m³ ml^–1). The feeding rate data best fit an Ivlev feeding function. An autoallelopath might be causing the repression. Water preconditioned with crowded Daphnia completely repressed feeding in uncrowded Daphnia after six hours.     

Initiation and growth characteristics of suspension cultures of Calendula officinalis cells

Callus cultures of marigold (Calendula officinalis L.) were induced on Murashige and Skoog medium with different concentrations of auxin (dichlorophenoxyacetic acid (2,4-D) or indole-3-acetic acid (IAA) and cytokinin (kinetin or 6-(,-dimethylallylamino)purine (2iP). Of all hormone combinations used in the medium, two were the most efficient in promoting callus development: 1.81 M (0.4 mg l^–1) 2,4-D and 1.85 M (0.4 mg l^–1) kinetin (0.4d–0.4k culture) or 0.45 M (0.1 mg l^–1) 2,4-D and 2.02 M (0.5 mg l^–1) 2iP (0.1d–0.5p culture). These combinations were selected to induce cell suspension cultures. The suspension cultures were maintained under light or dark conditions. The light stimulated cell aggregation in the cultures. In both cultures cells were undifferentiated under darkness, whereas in the light, rhyzogenesis was observed in 0.1d–0.5p culture. The cell growth and protein and oleanolic acid contents were determined. Initially, biomass production was similar under light and dark conditions, but after 7–8 months from the induction the cell growth was reduced by approximately 30% in the light, whereas the cell growth of the cultures maintained under darkness did not reveal any changes. The presence of oleanolic acid was detected in the suspension cultures kept in darkness. This compound reached two quantitative peaks: in the lag and stationary phases –- beyond the active growth phase of the culture cycle and its concentration was several times higher in 0.1d–0.5p culture than that in 0.4d–0.4k culture. It was for the first time that callus and suspension cultures were induced from the marigold plant.     

Infrared analysis of eleven carrageenophytes from Baja California,Mexico

Infrared analyses of the carrageenan in ten species (representing four genera) of Gigartinaceae and one species of Hypneaceae in different reproductive phases from the northwestern coast of Baja California were studied. Cystocarpic samples of the Gigartinaceae presented varying degrees of a / hybrid. The degree of hybridization was determined based on the ratio between the peak absorbances at 805/845 cm^–1. A high correlation was observed between the 805/845 cm^–1 and 805/970 cm^–1 ratios. Tetrasporic samples of Gigartina leptorhynchos, Iridaea splendens, Rhodoglossum affine and R. roseum, presented a -carrageenan profile, whereas Gigartina tepida, G. exasperata, G. harveyana, G. canaliculata and G. spinosa presented a -carrageenan. The tetrasporic sample of Hypnea valentiae showed a -carrageenan with a very low degree of hybridization.     

Voltage-gated chloride currents in cultured canine tracheal epithelial cells

Summary Chloride ions (Cl^–) are concentrated in airway epithelial cells and subsequently secreted into the tracheal lumen by downhill flux through apical Cl^– channels. We have studied Cl^– currents in cultured canine tracheal cells using the whole-cell voltage-clamp technique. Ultrastructural techniques demonstrated that the cells used in the electrophysiological experiments possessed apical membrane specializations known to be present in the intact, transporting cell type. Cultured cells 2–6 days old were characterized by an input resistance of 3.4±0.8 G (n=11) and a capacitance of 63.8±10.8 pF (n=26). A comparison of 3 and 4 day-old cells with 5 and 6 day-old cells showed that the input resistance decreased almost 50%, and the cell capacitance and the inward and outward currents increased concomitantly approximately 200%. Cultured cells 3–4 days old held at –40 mV produced currents of 196±22 pA at 50 mV and –246±27 pA at –90 mV (n=212) with pipette and bath solutions containing primarily 140 KCl and 140 NaCl, respectively. The chloride channel blocker diphenylamine-2-carboxylate (DPC, 100 m) suppressed whole-cell currents by 76.8% at 60 mV; however, currents were unaffected by the stilbenes SITS (1mm) and DNDS (1–30 m). Replacement of K⁺ with Cs⁺ in the pipette solution did not affect the outward current, the current reversal potential, or the input resistance of the cells, indicating that the current was not significantly K⁺ dependent when the intrapipette solution was buffered to a Ca²⁺ concentration of 20nm. The Cl^–/Na⁺ permeability ratio was estimated to be greater than 11 as calculated from reversal potential measurements in the presence of an internal to external NaCl concentration ratio of 12. Current equilibrium permeabilities, relative to Cl^– were: I^– (2.9)NO ₃ ^– (1.1)Br^– (1.1)Cl^– (1.0)F^– (0.93)MeSO ₄ ^– (0.19)gluconate (0.18)aspartate (0.14). Depolarizations to potentials greater than 20 mV elicited a time-dependent component in the outward current in 71% of the cells studied. Currents inactivated with a double exponential time course at the most depolarized voltages. Recovery from inactivation was fast, holding potential-dependent, and followed a double exponential time course. Current amplitude was increased via a cAMP-dependent pathway as has been demonstrated for single Cl-selective channels in cell-attached patches from cultured canine and human tracheal epithelial cells. Forskolin, an activator of adenylate cyclase, produced a 260% increase in the outward current at +50 mV. In summary, cultured canine tracheal cells have a single resting conductance that is Cl^– selective, voltage-dependent, and modulated by a cAMP-dependent mechanism. This preparation appears to be appropriate for analysis of cellular modulation of airway Cl^– channels and Cl^– secretion.     

Changes in Photosystem II fluorescence in Chlamydomonas reinhardtii exposed to increasing levels of irradiance in relationship to the photosynthetic response to light

The effects of a 60 min exposure to photosynthetic photon flux densities ranging from 300 to 2200 mol m^–2s^–1 on the photosynthetic light response curve and on PS II heterogeneity as reflected in chlorophyll a fluorescence were investigated using the unicellular green alga Chlamydomonas reinhardtii. It was established that exposure to high light acts at three different regulatory or inhibitory levels; 1) regulation occurs from 300 to 780 mol m^–2s^–1 where total amount of PS II centers and the shape of the light response curve is not significantly changed, 2) a first photoinhibitory range above 780 up to 1600 mol m^–2s^–1 where a progressive inhibition of the quantum yield and the rate of bending (convexity) of the light response curve can be related to the loss of Q_B-reducing centers and 3) a second photoinhibitory range above 1600 mol m^–2s^–1 where the rate of light saturated photosynthesis also decreases and convexity reaches zero. This was related to a particularly large decrease in PS II centers and a large increase in spill-over in energy to PS I.Abbreviations Chl chlorophyll - DCMU 3,(3,4-dichlorophenyl)-1,1-dimethylurea - F_M maximal fluorescence yield - F_pl intermediate fluorescence yield plateau level - F₀ non-variable fluorescence yield - F_v total variable fluorescence yield (F_M-F₀) - initial slope to the light response curve, used as an estimate of initial quantum yield - convexity (rate of bending) of the light response curve of photosynthesis - LHC light-harvesting complex - P_max maximum rate of photosynthesis - PQ plastoquinone - Q photosynthetically active photon flux density (400–700 nm, mol m^–2s^–1) - PS photosystem - Q_A and Q_B primary and secondary quinone electron acceptor of PS II     

Isospora lutrae n. sp. (Apicomplexa: Eimeriidae), a new coccidium from the European otter Lutra lutra (L.) (Carnivora: Mustelidae) from Spain

Parasitological examination of European otter originating from Extremadura, Spain revealed the presence of a new isosporan species. Oöcysts of Isospora lutrae n. sp. are spherical to subspherical, 31.2 (27.5–32) × 29.6 (28–31) m and have a smooth wall c. 1 m thick. Sporocysts are ellipsoidal, 18.2 (17–19) × 14.4 (14–16) m and lack Stieda and substieda bodies. A spherical sporocyst residuum is present, consisting of granules scattered among the sporozoites. Sporozoites are spindle-shaped, 12.4 × 2.5 m and have anterior and posterior refractile bodies. Based on its unique morphologic structure and host, I. lutrae is considered to be new.     

Fine-scale movement and assimilation of carbon in saltmarsh and mangrove habitat by resident animals

Despite theories of large-scale movement and assimilation of carbon in estuaries, recent evidence suggests that in some estuaries much more limited exchange occurs. We measured the fine-scale movement and assimilation of carbon by resident macroinvertebrates between adjacent saltmarsh and mangrove habitats in an Australian estuary using ¹³C analysis of animals at different distances into adjacent patches of habitat. ¹³C values of crabs (Parasesarma erythrodactyla –15.7 ± 0.1, Australoplax tridentata –14.7 ± 0.1) and slugs (Onchidina australis –16.2 ± 0.3) in saltmarsh closely matched that of the salt couch grass Sporobolus virginicus (–15.5 ± 0.1). In mangroves, ¹³C values of crabs (P. erythrodactyla –22.0 ± 0.2, A. tridentata –19.2 ± 0.3) and slugs (–19.7 ± 0.3) were enriched relative to those of mangroves (–27.9 ± 0.2) but were more similar to those of microphytobenthos (–23.7 ± 0.3). The ¹³C values of animals across the saltmarsh-mangrove interface fitted a sigmoidal curve, with a transition zone of rapidly changing values at the saltmarsh-mangrove boundary. The width of this transition indicated that the movement and assimilation of carbon is limited to between 5 and 7 m. The ¹³C values of crabs and slugs, especially those in saltmarsh habitat, clearly indicate that the movement and assimilation of carbon between adjacent saltmarsh and mangrove habitat is restricted to just a few metres, although some contribution from unmeasured sources elsewhere in the estuary is possible. Such evidence demonstrating the extent of carbon movement and assimilation by animals in estuarine habitats is useful in determining the spatial arrangement of habitats needed in marine protected areas to capture food web processes.     

Gonadal neoplasms in wild carp-goldfish hybrids from the Welland River near Niagara Falls,Canada

An experimental approach was taken to examine the processes of detritus decomposition in river sediments.Addition of macrophyte detritus (Alternanthera philoxeroides (Mart.) Griseb) to river sediment resultedin an increase in carbon mineralization from a sbasal rate of 200 up to 500 mg C m^–2 d^–1.Carbon mineralization after addition of oak detritus was only slightly higher than mineralization in sediments thatreceived no addition ( 200 mg C m^–2 d^–1). Bacterial biomass and production in sediments \s+ alligator-weedwere higher than in sediments + oak or zero-addition. In these experiments the major fate of addedalligatorweed was mineralization. For alligatorweed detritus, microbial metabolism depletes organic carbonrather than leading to increases in food quality. Therefore, a pulse input of alligatorweed detritus would notbe available as a long-term source of organic carbon. Oak detritus was not rapidly decomposed, and sopersists in these sediments for a longer period.     

Pheromone puffs suppress mating by Plodia interpunctella and Sitotroga cerealella in an infested corn store

The efficacy of pheromone mating disruption was investigated in a 7×6×3 m corn storage room harboring a high population density of Indian meal moth, Plodia interpunctella (Hübner) and Angoumois grain moth, Sitotroga cerealella (Olivier). Pheromones were released from a controlled release dispenser, the metered semiochemical timed release system (MSTRS^TM) at emission rates of 0.6 g min^–1 (Z9,E12:14:Ac for Indian meal moth) and 0.2 g min^–1 (Z7,E11-16:Ac for Angouimois grain moth). Mating disruption efficacy was evaluated using three parameters: male capture in pheromone traps, visual examination of mating behavior, and the incidence and frequency of mating as measured by spermatophores. In three trials, comparisons were made between data collected before pheromone treatment and during treatment. Disruption of pheromone source location by males averaged 70% and 40% for P. interpunctella and S. cerealella, respectively, in the three trials. In addition, reduced levels of copulation by both species were recorded during pheromone treatment. More importantly, significant reductions were recorded in the incidence and frequency of mating by females of both species collected during the treatment period. While 85% of P. interpunctella females collected before pheromone treatment in three trials had mated at least once, only 50% of the females collected during treatment had mated. The mean number of matings, as measured by spermatophores, ranged between 0.8–1.1 and 0.5–0.7 before and during pheromone treatment, respectively. Similarly, a 20–30% reduction in the proportion of mated S. cerealella females was recorded during pheromone treatment. In the three trials, mean number of spermatophores per S. cerealella female averaged 1.0 and 0.7 during the pretreatment and treatment periods, respectively. Additional tests conducted in small boxes also recorded significant mating disruption of both species.     

Isolation of membrane bound light-harvesting-complexes from the dinoflagellates Heterocapsa pygmaea and Prorocentrum minimum

We have isolated Chl a-Chl c-carotenoid binding proteins from the dinoflagellates Prorocentrum minimum and Heterocapsa pygmaea grown under high (500 mol m^–2 s^–1, HL) and low (35 mol m^–2 s^–1, LL) light conditions. We compared various isolation procedures of membrane bound light harvesting complexes (LHCs) and assayed the functionality of the solubilized proteins by determining the energy transfer efficiency from the accessory pigments to Chl a by means of fluorescence excitation spectra. The identity of the newly isolated protein-complexes were confirmed by immunological cross-reactions with antibodies raised against the previously described membrane bound Chl a-c proteins (Boczar et al. (1980) FEBS Lett 120: 243–247). Spectroscopic analysis demonstrated the relatedness of these proteins with the recently described Chl-a-c ₂-peridinin (ACP) binding protein (Hiller et al. (1993) Photochem Photobiol 57: 125–131; Iglesias Prieto et al. (1993) Phil Trans R Soc London B 338: 381–392). The water-soluble peridinin-Chl a binding-protein (PCP) was not detectable in P. minimum. Two functional forms of ACP with different pigmentation were isolated. A variant of ACP which was isolated from high-light grown cells, that specifically binds increased amounts of diadinoxanthin was compared to the previously described ACPs that bind proportionately more peridinin.Abbreviations ACP Chl a-Chl c-peridinin binding protein - AEBSF 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride - DDM dodecyl -d maltoside - Deriphat 160 N-lauryl-beta-iminopropionic acid - HEPES (N-2-hydroxyethylpiparizine-N-2-ethanesulphonic acid) - HL high light (500 mol m^–2 s^–1) - LL low light (35 mol m^–2 s^–1) - ₇₃₀ fluorescence yield (emission at 730 nm) - PCP peridinin-Chl a-binding protein - PMSF phenyl-methyl-sulfonyl-fluoride - PS I Photosystem I - PS II Photosystem II     

Limitations associated with conductivity measurement for monitoring growth in plant tissue culture

The suitability of conductivity measurement for monitoring growth in plant cell culture has been tested using suspended cells and genetically-transformed hairy roots of Atropa belladonna, and aggregated cells of Solanum aviculare. Other researchers have proposed that a constant ratio exists between increase in cell concentration (x) and decrease in medium conductivity (C). In all cases studied in this work, x/C was not constant over a wide range of cell densities tested in batch culture. With cell suspensions, x/C decreased continuously during the growth phase from 3.4 to 2.5 g cm l^–1 mS^–1. For the hairy roots, the ratio between x and C varied by as much as 4-fold during growth. The relationship between conductivity and growth for S. aviculare aggregates was found to vary depending on inoculum density. No simple correlation between conductivity change and cell growth was apparent for the plant-cell systems studied.     

Effect of laminar flow velocity on the kinetics of surface recolonization by Mot+ and Mot− Pseudomonas fluorescens

Computer-enhanced microscopy (CEM) was used to monitor bacteria colonizing the inner surfaces of a 1×3 mm glass flow cell. Image analysis provided a rapid and reliable means of measuring microcolony count, microcolony area, and cell motility. The kinetics of motile and nonmotilePseudomonas fluorescens surface colonization were compared at flow velocities above (120m sec^–1) and below (8m sec^–1) the strain's maximum motility rate (85m sec^–1). A direct attachment assay confirmed that flagellated cells undergo initial attachment more rapidly than nonflagellated cells at high and low flow. During continuous-flow slide culture, neither the rate of growth nor the timing of recolonization (cell redistribution within surface microenvironments) were influenced by flow rate or motility. However, the amount of reattachment of recolonizing cells was both flow and motility dependent. At 8m sec^–1 flow, motility increased reattachment sixfold, whereas at 120m sec^–1 flow, motility increased reattachment fourfold. The spatial distribution of recolonizing cells was also influenced by motility. Motile cells dispersed over surfaces more uniformly (mean distance to the nearest neighbor was 47.0m) than nonmotile cells (mean distance was 14.2m) allowing uniform biofilm development through more effective redistribution of cells over the surface during recolonization. In addition, motile cell backgrowth (where cells colonize against laminar flow) occurred four times more rapidly than nonmotile cell backgrowth at low flow (where rate of motility exceeded flow), and twice as rapidly at high flow (where flow exceeded the rate of motility). The observed backgrowth of Mot⁺ cells against high flow could only have occurred as the result of motile attachment behavior. These results confirm the importance of motility as a behavioral mechanism in colonization and provides an explanation for enhanced colonization by motile cells in environments lacking concentration gradients necessary for chemotactic behavior.     

Fabrication of Stable Packed Capillary Reversed-Phase Columns for Protein Structural Analysis

Capillary column (320-m ID) liquid chromatography is an essential tool for the separation and concentration of low-picomole amounts of proteins and peptides for mass-spectrometric based structural analysis. We describe a detailed procedure for the fabrication of stable and efficient 50- to 180-m ID polyimide fused-silica columns. Columns were packed by conventional slurry packing with reversed-phase silica-based supports followed by column bed consolidation with acetonitrile and sonication. PVDF membrane or internal fused-silica particles were employed for column end-frit construction. The ability of these columns to withstand high back pressures (300–400 bar) enabled their use for rapid chromatography (>3400 cm/hr; i.e., 40 l/min for 200-m ID columns) and the loading of large sample volumes (up to 500 l). The accurate low flow rates (0.4–4.0 l/min) and precise gradient formation necessary to operate these columns were achieved by a simple modification of conventional HPLC systems [Moritz et al. (1992), J. Chromatogr. 599, 119–130]. Column performance was evaluated for ability to resolve low-fmol amounts of all components of a mixture of PTH-amino acids and to separate peptides for on-line LC/MS analysis of peptide mixtures derived from in situ digestion of 2-DE resolved protein spots.     

Abiotic metal stress enhances diosgenin yield in Dioscorea bulbifera L. cultures

Lower concentrations of CuSO₄ (25–75 M) in the MS medium supplemented with 0.1 mg l^–1 IAA+5.0 mg l^–1 Kn+500 mg l^–1 CH+10 mg l^–1 Cyst hyd enhanced the growth of regenerants of Dioscorea bulbifera L. CuSO₄ (75 M) induced an appreciable diosgenin yield in the regenerants compared to those obtained on media without Cu. The presence of Cu thus seems to stimulate diosgenin production. The regenerants also differentiated bulbils on lower concentrations of Cu. At CuSO₄ (100 M), however, cultures showed poor growth as well as a low diosgenin yield. Increased proline and protein contents were recorded in cultures grown on Cu-enriched media.