Secretion of β-lactamase by Escherichia coli in vivo and in vitro: effect of cerulenin

The effect of cerulenin on the production of -lactamase and other periplasmic proteins was studied in Escherichia coli IA199 carrying plasmid pBR322. Cerulenin (10 to 25 g/ml) had almost no effect on the growth rate of E. coli but it decreased the amount of -lactamase and other periplamic proteins in shock fluid. Higher amounts of the antibiotic (40 to 100 g/ml)decreased turbidity and almost completely prevented synthesis of -lactamase and other periplasmic proteins. Cerulenin decreased incorporation of l-[³⁵S]methionine into membranes during growth as well. Spheroplasts secreted -lactamase into the external medium, but during a 3-h incubation in the presence of cerulenin (25 g/ml) this secretion was prevented by more than 90%. -Lactamase was secreted into the isolated membrane vesicles from E. coli IA199. However, only 5% of the total amount of pre--lactamase was secreted and processed by the membranes in vitro. Cerulenin did not prevent processing in vitro but the membranes prepared from the cells grown in the presence of cerulenin (25 g/ml) did not catalyze processing of pre--lactamase at all. Membrane preparations from Bacillus subtilis did not process pre--lactamase either in the absence or in the presence of cerulenin.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Coral growth in high-nutrient,low-pH seawater: a case study of corals cultured at the Waikiki Aquarium,Honolulu, Hawaii

Fifty-seven species of hermatypic corals have been maintained and grown in high-nutrient seawater at the Waikiki Aquarium, Honolulu, Hawaii. In this study we document the chemical conditions of aquarium water in terms of dissolved nutrients and carbon. Aquarium water is characterized by concentrations of inorganic nutrients that are high relative to most natural reef ecosystems: SiO₃ 200 M; PO₄ 0.6 M; NO₃ 5 M; NH₄ 2 M. In contrast, concentrations of organic nutrients are lower than most tropical surface ocean waters: DOP 0.1 M and DON 4 M. The incoming well-water servicing the facility has low pH, crating over-saturation of carbon dioxide. The coral communities in aquaria took up inorganic nutrients and released organic nutrients. Rates of nutrient uptake into aquaria coral communities were similar to nutrient uptake by natural reef communities. Coral growth rates were near the upper rates reported from the field, demonstrating corals can and do flourish in relatively high-nutrient water. The growth of corals does not appear to be inhibited at concentrations of nitrogen up to 5 M. Statements implying that corals can only grow in low nutrient oligotrophic seawater are therefore oversimplifications of processes that govern growth of these organisms. Some basic guidelines are given for maintenance of coral communities in aquaria.     

Mathematical modelling for the generation of L-[3-2H,3-13C]lactic acid isotopomers by erythrocytes exposed to either D-[1-13C]glucose or D-[6-13C]glucose in the presence of2H2O

Cytosolic purine nucleoside phosphorylase (PNPase) is a well known, and described enzyme which exists in a variety of organisms, both procaryotic and eucaryotic. More recently this enzyme was found in bovine liver mitochondria. The mitochondrial purine nucleoside phosphorylase was purified 63 fold and has a molecular weight of 48–60 kD. From Lineweaver-Burk plots apparent K_m's of 23M for inosine, 42 M for deoxyinosine, 40 M for phosphate, 2 M for hypoxanthine, and 163 M for ribose-1-phosphate were calculated. Both 8-aminoguanosine (K_i=0.5 M) and araG (K_i=381 M) are inhibitors of the enzyme. The protein's isoelectric point (pI) was calculated at a pH of 4.2. Preliminary immunological work showed no cross-reactivity between epitopes on the mitochondrial protein and those on PNPase from human erythrocytes. The apparent K_m's calculated for the mitochondrial enzyme are,with the exception of that using hypoxanthine, within the range commonly associated with K_m's from the cytosolic species. The mitochondrial enzyme's molecular weight and pI are less than normally described. The enzyme's isolation from mitochondria, together with several unique characteristics, suggest that it is a separate protein from that found in the cytosol.     

The spermatozoon of Oikopleura dioica Fol (Larvacea,Tunicata)

Summary The spermatozoon of Oikopleura dioica is about 30 m long, with a spherical head, about 1 m wide, a 3 m long and 1 m wide midpiece, and a 25 m long tail with a tapered end piece. The head contains a nucleus with the chromatin volume limited to about 0.1 m³. A small acrosome is found in an anterior inpocketing, and a flagellar basal body in a posterior inpocketing of the nucleus. The midpiece contains a single mitochondrion with the flagellar axoneme embedded in a groove along its medial surface. The flagellar axoneme has the typical 9 + 2 substructure, and the basal body the typical 9+0 substructure. A second centriole and special anchoring fibres are absent.     

Fouling of ceramic membranes during microfiltration of model solutions

A model study of the microfiltration of fatty acids (C2 to C8) dissolved in a synthetic ultrafiltrate was performed. Ceramic membranes of 0.1 and 0.2 m were used. Results showed that minerals from the ultrafiltrate were involved in membrane fouling, while fatty acids were poorly rejected.On 0.1 m membranes, modelisation of fouling exhibited two different steps of fouling, a first one due to adsorption of solutes, and a second one involving particles deposit onto the membrane. On 0.2 m membranes, only fouling due to particles deposit was observed in modelisation. Results of fouling and rejection pointed out the great difference between 0.1 and0.2 m membranes.This revised version was published online in October 2005 with corrections to the Cover Date.     

Influence of nitrogen on the behaviour of nickel in wheat

A glasshouse experiment was conducted to study the effect of Ni on the growth and nutrients concentration in wheat (Triticum aestivum Cv. WH 291) in the presence and absence of applied N as urea. Responses to N application were observed up to 120 g N g^–1 soil. No response to Ni was observed in the dry matter yield of wheat tops (leaves + stem) in the absence of applied N while in the presence of applied N, significant yield increases were obtained at 12.5g Ni g^–1 soil. Nickel was not toxic to wheat up to 50g Ni g^–1 soil in the presence of 120g N g^–1 soil. Nitrogen and Ni concentration in wheat tops and roots increased with increasing levels of applied N and Ni, respectively. Applied Ni had an antagonistic effect on N concentration. Similarly, N reduced the Ni concentration in the wheat tissues. Positive growth responses to Ni were associated with 22 and 15g Ni g^–1 in wheat tops, in the presence of applied N at 60 and 120g N g^–1 soil, while Ni toxicity was associated with 63, 92.5 and 112.5g Ni g^–1 in wheat tops, in the absence and presence of applied N at 60 and 120g N g^–1 soil, respectively.     

6-Methylpurine-induced inhibition of sclerotia morphogenesis in Sclerotium rolfsii and its reversal by adenosine

In liquid synthetic medium inoculated with Sclerotium rolfsii (SR), addition of 6-methylpurine (MP, 50g/ml) immediately after inoculation led to approximately 100% reduction in sclerotia production. Adenosine, and to a lesser extent guanosine, each at final concentration of 100g/ml significantly reduced inhibition of sclerotia formation by SR in presence of 50g/ml MP. Uridine and cytidine each at 100g/ml had no such effect. The inhibition of sclerotia morphogenesis could be prevented by addition of 800g/ml of adenosine together with 50g/ml MP. Reversal by adenosine of MP-induced inhibition of sclerotia development was concentration dependent.     

The contribution of size-fractionated plankton to biomass and primary production of phytoplankton in saline–alkaline ponds

Primary productivity, biomass and chlorophyll-a of size fractionated phytoplankton (<0.22 m, <3 m, <8 m, <10 m, <40 m, <64 m, <112 m and <200 m) were estimated in 6 ponds and 5 experimental enclosures. The results showed that the planktonic algae less than 10 m are important in the biomass and production of phytoplankton in saline–alkaline ponds. The production of size fractionated phytoplankton corresponding to <112 m, <10 m and <3 m in saline–alkaline ponds were 10.5 ± 6.6 , 8.6 ± 5.4 and 0.33 ± 0.1 mgC l^–1 d^–1, respectively. Mean community respiration rate was 1.80 ± 0.73, 1.69 ± 0.90 and 1.38 ± 1.12 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) were 1.61, 8.30 and 0.33 mgC l^–1 d^–1, respectively. The ratio of those to the total phytoplankton production was 15%, 79% and 3%, respectively. The mean respiration rate of the different size groups was 0.11, 0.31 and 1.38 mgC l^–1 d^–1; the ratio of those to total respiration of phytoplankton was 6%, 17% and 77%, respectively. The production of size-fractionated phytoplankton corresponding to <200 m, <10 m and <3 m in enclosures was 2.19 ± 1.63, 2.08 ± 1.75 and 0.22 ± 0.08 mgC l^–1 d^-1, respectively. Mean community respiration rates were 1.25 ± 1.55, 1.17 ± 1.42 and 0.47 ± 0.32 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton was 0.11, 1.86 and 0.22 mgC l^–1 d^–1, respectively. The ratio of those to the total production of phytoplankton was 5%, 85% and 10%, respectively. The mean respiration rate of different size groups were 0.08, 0.72 and 0.46 mgC l^–1 d^–1, the ratio of those to total respiration of phytoplankton was 6%, 57% and 37%, respectively. The concentrations of chlorophyll-a of the phytoplankton in the corresponding size of micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) plankton in the experimental ponds were 19.3, 98.2 and 11. 9 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 15%, 76% and 9%, respectively. The concentrations of chlorophyll-a of phytoplankton micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton in enclosures were 1.7, 34.3 and 3.0 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 4%, 88% and 8%, respectively.     

Influence of sarmesin on some dopamine-related types of behaviour

Summary The present study was undertaken to investigate the effects of sarmesin, an analogue of [Sar¹] angiotensin II (ANG II) where the tyrosine hydroxyl group in position 4 is methylated, on dopamine (DA)-related paradigms: locomotor and exploratory behaviour as well as apomorphine (3 mg/kg, ip)-induced stereotypy in rats. Sarmesin (0.5 and 1 g, icv) significantly decreased ambulation and rearing movements, and blocked the inhibitory effect of ANG II (0.1 g) on both types of activity. Sarmesin induced biphasic effects on apomorphine-induced stereotypy depending on the dose increase (0.5 and 5 g, icv) and decrease (10 g). Moreover, sarmesin (5 g) blocked the inhibitory effect of ANG II (2 g, icv) on apomorphine stereotypy. Taken together, these results suggest that sarmesin might interact with AT₁ and AT₂ receptor subtypes. The results further confirm the statement for ANG II-DA interaction in brain structures involved in these types of behaviour.     

Adventitious shoot regeneration from in vitro-cultured leaves of Rubus genotypes

Regeneration of adventitious shoots from leaves of several in vitro-cultured Rubus genotypes was affected by media components and incubation conditions. Leaves cultured at 20°C with a photosynthetic photon flux (PPF) of 40 mol m^-2 s^-1 had a higher regeneration frequency and more shoots per regenerating leaf than ones cultured at 25°C with a PPF of either 40 or 80 mol m^-2 s^-1. Thidiazuron (TDZ) was significantly more effective than benzyladenine. Medium containing 1 M TDZ had the highest percentage regeneration for leaves of Autumn Bliss, Canby, Summit and Sentry red raspberries, whereas leaves of MD-ETCE-1 blackberry hybrid responded more to 10 M TDZ. Higher regeneration frequencies were obtained with 0.5 M indolebutyric acid (IBA) than with 1 M, but no significant difference was observed between 0.5 M and no IBA in other experiments. More shoots per regenerating leaf formed on Murashige and Skoog (MS) and N6 media, than on half-strength MS, Anderson, or Woody Plant media for all genotypes tested. The youngest two expanding leaves near the shoot apex were the most regenerative and yielded the highest number of shoots per regenerating leaf.Abbreviations AND Anderson (1980) - BA benzyladenine - IBA indolebutyric acid - MS Murashige & Skoog (1962) - N6 Chu et al. (1975) - NAA naphthaleneacetic acid - PPF photosynthetic photon flux - TDZ thidiazuron (N-phenyl-N'-1, 2, 3-thiadiazol-5-ylurea) - WPM Woody Plant Medium [Lloyd & McCown (1980)]     

Method of isolation of cysteine constitutive mutants of the cysteine regulon in Salmonella typhimurium.

Summary Size and shape of mitochondrial DNA molecules of Schizosaccharomyces pombe were analyzed by electron microscopy. Besides numerous linear molecules, circular molecules ranging from 0.83 m to 12.81 m were found. Depending on the method of preparation, both closed and open circular molecules were found. Most of the circular molecules could be assigned to five major size classes of 0.83±0.05 m, 1.7±0.05 m, 4.74±0.04 m, 5.74±0.04 m, and 8.32±0.07 m. Possible explanations for the different size classes of mitochondrial DNA molecules are discussed.     

The influence of membrane potential on chloride channels activated by GABA in rat cultured hippocampal neurons

Chloride currents were activated by a low concentration of GABA (0.5 m) in neonatal rat hippocampal neurons cultured for up to 14 days. Currents elicited by 0.5 m GABA in neurons, voltage-clamped using the whole-cell technique with pipettes containing 149 mm Cl^–, reversed close to 0 mV whether pipettes contained 144 mm Na⁺ or 140 mm Cs⁺, and were blocked by 100 m bicuculline. Current-voltage curves showed outward rectification. Single channel currents appeared in cell-attached patches when the pipette tip was perfused with pipette solution containing 0.5 m GABA and disappeared when a solution containing 100 m bicuculline plus 0.5 m GABA was injected into the pipette tip. The channels showed outward rectification and, in some patches, had a much lower probability of opening at hyperpolarized potentials. The average chord conductance in 10 patches hyperpolarized by 80 mV was 7.8±1.6 pS (sem) compared with a chord conductance of 34.1±3.5 pS (sem) in the same patches depolarized by 80 mV. Similar single channel currents were also activated in cell-free, inside-out patches in symmetrical chloride solutions when 0.5 m GABA was injected into the pipette tip. The channels showed outward rectification similar to that seen in cell-attached patches, and some channels had a lower probability of opening at hyperpolarized potentials. The average chord conductance in 13 patches hyperpolarized by 80 mV was 11.8±2.3 pS (sem) compared with 42.1±3.1 pS (sem) in the same patches depolarized by 80 mV.We are grateful to B. McLachlan and M. Robertson for their general assistance, to C. McCulloch and M. Smith for writing computer programs and to W. O'Hare for making the pipette injection device.     

Aflatoxin influences on seed germination and root elongation by two cultivars of Glycine max and uptake of 65 Zn-ZnCl2 by the cultivars

Maximum inhibition of Glycine max, cv. Essex seed germination occurred at 10 g/ml following 72 hr imbibition in constant light. Seeds imbided 108 hr in constant darkness at this concentration showed a 20% rise in germination over that of the control. Imbibition of G. max, cv. Williams seeds in either light or dark for 96 hr did not suppress germination. Imbibition of Essex seeds in either light or dark at 2.5 through 10 g/ml stimulated root elongation except for 10 g/ml at 96 hr (light). Maximum inhibition of Williams root elongation under constant light was at 48 and 72 hr with 10 g/ml. Statistically significant differences in cotyledon, leaf and stem lengths between non-treated (NT) and treated (T) seedlings were not found except for Williams stem length at 2.5 / ml. Root elongation was stimulated 1.2- and 1.1-folds, respectively, at 5.0 (Essex) and 2.5 (Williams) g/ml. Toxin at 2.5 through 10.0 g/ml did not markedly alter either cotyledon or leaf widths with the exception of Williams leaf width at 2.5 g/ml. Medium supplementation with 2.5 through 10.0 g/ml resulted in cotyledon, leaf and root weight enhancements for Essex seedlings. Stem weight was not markedly affected. An 18% rise in Williams cotyledon weight above that of the control was seen at 2.5 g/ml. Williams leaf weights were increased 1.75- and 1.25-folds, respectively, at 2.5 and 10.0 g/ml. Aflatoxin B₁, at 2.5 g/ml promoted Williams stem and root elongation 1.20- and 1.09-folds, respectively. Most of the radioactivity from ⁶⁵Zn-ZnCl₂ recovered within organs was found within Essex roots for both T and NT seedlings. A higher amount of radioactivity was recovered within roots at each toxin concentration than was without toxin. However, this was not statistically significant. Significant differences in the distribution of radioactivity within roots between NT and T Williams seedlings were not observed. Generally, AFB₁ failed to affect significantly these two varieties of soybeans based on the tests relating to germination, growth and radiolabel uptake.     

Problems in estimating growth rates of marine phytoplankton from short-term14C assays

Growth rate estimates () of phytoplankton populations that were sampled from nitrogen-limited continuous cultures and then incubated for short durations in batch culture with added¹⁴C-HCO₃ ^– were significantly different than steady-state growth rates () for 3 of 5 marine phytoplankton species. Two diatoms,Thalassiosira weissflogii andChaetoceros simplex, displayed virtually identical growth rates (=) over a wide range of, whereas for a third diatom,Phaeodactylum tricornutum, was overestimated by an average of 40% compared to. In contrast, was underestimated by the¹⁴C technique for the two remaining species: up to 40% at a steady-state of 1.0 day^–1 for the chlorophyteDunaliella tertiolecta and up to 100% at of 1.4 day^–1 for the haptophytePavlova lutheri. For the latter two species the divergence between and appeared to increase with increasing steady-state. A simple model of labeled and total carbon flow between the aqueous phase and cellular biomass was constructed to demonstrate that respiration was negligible when=, but was significant when>. In the cases in which<, a rapid physiological alteration presumably took place once the steady state was disturbed and cells were placed in the incubation chambers, which perhaps was related to the nutritional state of the cultures at the time of sampling. Questions thus are raised regarding our ability to measure accurately primary productivity from shipboard experiments with confined samples of phytoplankton from nutrient-impoverished waters that probably are less hardy than the laboratory cultures used in these studies.     

Artemisia annua L.: dedifferentiated and differentiated cultures

Dedifferentiated and differentiated tissue cultures ofArtemisia annua L. for artemisinin production were carried out. The calluses were initiated on MS medium supplemented with sucrose (30 g l^-1), myoinositol (100 mg l^-1) and RT vitamins. The auxins used were naphtaleneacetic acid (NAA), indoleacetic acid (IAA), indolebutyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-d). These were added to the basal medium either singly or in combination. The best results were obtained with 2.4-d (4.5 M : 0.02 d^-1) and NAA (5.4 M : 0.06 d^-1). Cell suspensions were established on the same media without agar. Suspension cultures showed different morphological characteristics according to the plant growth regulator supplied. Organized cultures were initiated from callus obtained on 2,4-d (4.5 M) and from bud cultures. Medium containing 6-benzylaminepurine (BA) (8.9 M)+NAA (0.54 M); Zeatin (45.62 M)+NAA (5.37 M) or BA (8.9 M) stimulated both organogenesis in callus (frequency of induction =50%) and semi-organized tissue in shoot buds. BA (13.32 M)+NAA (1.08 M) or BA (13.32 M) only stimulated multiple shoot cultures (frequency of induction =80%). Regarding artemisinin content, while the values obtained were 1.13 and 0.78 mg gDW^-1 in primary callus, artemisinin was not detected in cell suspension and only traces of it were found in multiple shoot cultures.     

Replicating DNA molecules from eggs ofDrosophila melanogaster

Eggs ofDrosophila melanogaster were lysed with sodium dodecyl sulphate within 110 minutes after laying and the lysate prepared for electron microscopy by the protein monolayer technique. Long, non-circular DNA molecules were found with a form suggesting they contained either a single replicated region, or two, three or four replicated regions arranged in tandem. Each replicated region was delimited by two forks. The two segments of DNA spanning the region between the forks were approximately equal in length and appeared to be totally or almost totally double-stranded. The appearance of replicating molecules was not altered by digestion with pronase or treatment with phenol or chloroform. The lengths of replicated regions varied from 0.2 to 22.1 with a mean value of 2.97 . The distances between midpoints of adjacent tandemly arranged replicated regions ranged from 1.2 to 9.7 with a mean value of 3.87 . Circular molecules found in these preparations, and presumed to be of mitochondrial origin, were estimated from comparative length measurements with circular double-stranded DNA molecules from the bacteriophages lambda, X174 and fd to have a molecular weight of 12.36 X 10⁶ daltons.     

Neurotoxic Potential of Three Structural Analogs of β-N-oxalyl-α,β-Diaminopropanoic Acid (β-ODAP)

Lathyrism is a non-progressive motor neuron disease produced by consumption of the excitatory amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (-ODAP). To learn more about the mechanisms underlying Lathyrism three structural analogs of -ODAP were synthesized. Carboxymethyl-,-diaminopropanoic acid (CMDAP) evoked inward currents which were antagonized by APV (30 M), but not by CNQX (10 M). N-acetyl-,-diaminopropanoic acid (ADAP) evoked no detectable ionic currents but potentiated N-methyl-D-aspartate (NMDA)-activated currents. The potentiation of NMDA currents by ADAP was blocked by 7-chlorokynurenic acid. Carboxymethylcysteine (CMC) did not activate any detectable ionic currents. None of the three -ODAP analogs produced visible symptoms of toxicity in day old chicks when administered for 2–3 consecutive days. Ligand binding studies demonstrated that all the three compounds were effective to in displacing [³H]glutamate. The maximum inhibition was 92% for CMDAP, 61% for ADAP, 65% for CMC and 99% for -ODAP. These data indicate that analogs of -ODAP may interact with glutamate receptors without producing neurotoxicity.     

Effects of lead and cadmium on chemical composition and total water content of the pupal parasitoid, Pimpla turionellae

The parasitoid Pimpla turionellae L. (Hymenoptera, Ichneumonidae) was fed on Cd, Pb and Cd+Pb-contaminated food (33g Cd, 82g Pb and 33g Cd+82g Pb per gram food fresh weight, respectively). Significant decrease in the total lipid and protein content was found along with an increase in the water content particularly in Cd-contaminated parasitoids.     

Slow growth phenotype - A possible approach to improved plasmid maintenance in Saccharomyces cerevisiae

Summary Transformation-induced slow growth phenotype (SGP) in yeast is repressed in the presence of 2m plasmids. A full 2m-sequence-based recombinant plasmid (pJB502) was found to be more stable in a 2m-free- [cir] strain of Saccharomyces cerevisiae than in a cir⁺ strain. This could not be attributed to differences in growth rate calculated from kinetic analysis of plasmid loss, but transformed [cir] isolates, which had lost the recombinant plasmid, exhibited varying degrees of SGP in batch culture. One of these isolates was outcompeted in chemostat culture by the recombinant-plasmid-containing strain, suggesting that improved plasmid maintenance can result from SGP in cir hosts.