Metabolic responses of microbiota to diesel fuel addition in vegetated soil

The effects of trees and contamination on microbial metabolic activity, especially that of hydrocarbon degrading bacteria, were compared during phytoremediation to find which conditions increase diesel fuel removal. Diesel fuel utilisation, microbial extracellular enzyme activities and utilisation of Biolog ECO plate carbon sources by soil bacteria were determined during phytoremediation experiments consisting of two separate diesel applications. Diesel fuel removal after 28 days of second diesel application was 20–30% more than after the first application 1 year earlier. Soil microbiota utilised 26–31 of the 31 Biolog ECO plate carbon sources. Carbon source utilisation profiles indicated minor differences in microbiota in soil vegetated with pine compared to microbiota in soil vegetated with poplar. The potential maximum rates of aminopeptidase activity were 10–10² M AMC/h/g dry soil prior to and after second diesel application, except 14days after the second diesel addition, where the rates were at the scale of 10³M AMC/h/g dry soil. The potential maximum rates of esterase activity were 10³–10⁴M MUF/h/g dry soil. The presence of plants did not influence the activity of esterases. The utilisation of diesel by soil bacteria in Biolog MT2 plate assay was higher in contaminated soil, especially when vegetated, than in uncontaminated soil, measured both as lag times and maximum specific utilisation rates. MT2 plate assay detected the biological response after diesel fuel addition better than general activity methods.     

The quantitative analysis of chromosome pairing and chiasma formation based on the relative frequencies of M I configurations

Whilst reliable estimates of chiasma frequencies can usually not be obtained, the probability (b) of a chromosome arm to be bound by at least one chiasma can often be determined. In the absence of interference this probability equals (1–e ^–2), where 2 is the average chiasma frequency of the chromosome arm and the average crossover frequency or map length. In the presence of interference is shown to retain its genetic meaning as an additive metric that may describe the chromosome arm or other distinctive chromosome segment in terms of genetic recombination. It is a form of potential map length, comparable to, but numerically different from the regular map length. It is termed provisionally crossing-over potential.A chromosome with armsm andn with crossing-over potentials and will form ring bivalents with a frequency (1–e ^–2).(1–e ^–2); open bivalents with a frequency (1–e ^–2).e ^–2+(1–e ^–2).e ^–2; univalent pairs with a frequencye ^–2.e ^–2. Estimates of these frequencies yield equations from which and may be solved. In rye (Secale cereale) their ratio (q) is approximately two and differs from the mitotic arm length ratio of 1.4, indicating localization of chiasmata in the long arms.Graphs are given to show how, with constantq, the relation between the probabilitiesb _m andb _n of the two arms being bound changes with changing averageb.Data are presented on chiasma frequencies in M I, and compared with the frequencies expected in the absence of interference to give an impression of the degree of interference. Apparent fusion of chiasmata simulates interference.     

Die exoepithelialen Schleimdrüsenzellen von Arion empiricorum (Fér.)

Zusammenfassung Im Oberflächenepithel der Wegschnecke Arion empiricorum (Fér.) existieren drei Arten von exoepithelialen Schleimdrüsenzellen. Sie können auf Grund der Ultrastruktur, aber auch durch Heterochromasie bei Toluidinblaufärbung (bei verschiedenen pH-Werten), unterschieden werden. Der Typ der ventralen Sohlendrüse kommt in der Kriechsohle, der Schwanzdrüse, den Tentakeln, der Kopfhaut und in der vom Pneumostom ventrad ziehenden Flimmerrinne vor, der Typ der lateralen Sohlendrüse seitlich unter dem Sulcus lateralis. Die Manteldrüsen sind im übrigen Epithel zu finden.Die ventralen Sohlendrüsen sind durch ein eigenartig strukturiertes Ergastoplasma ausgezeichnet, die lateralen Sohlendrüsen besitzen ein Ergastoplasma, das auf Proteinbildung hinweist und die Manteldrüsen besitzen als Charakteristikum riesige Golgi-Vakuolen.

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Summary In the surface epithelium of the slug, Arion empiricorum (Fér.), there exist three types of exoepithelian slime-gland-cells. They can be distinguished on account of their ultrastructure as well as by the fact that they show heterochromatism at different pH-values after toluidine-blue-staining. The type of the ventrale Sohlendrüse occurs in the under surface of the foot, in the large slime-gland at the tail, in the tentacles, in the epidermis of the head and in the ciliated groove underneath the pneumostome, the type of the laterale Sohlendrüse on the sides underneath the sulcus lateralis. The Manteldrüsen are to be found in the other places of the epithelium.The ventrale Sohlendrüsen are characterized by their specific ultrastructure of the ergastoplasm, the laterale Sohlendrüsen by a type of ergastoplasm, which shows signs of protein-production, the Manteldrüsen however by containing huge Golgi-vacuoles.

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Herrn Professor L. Stockinger danke ich für Kritik und Ratschläge.     

Diagnosing lactic acid fermentation based on specific rates of growth,substrate consumption and product formation

The on-line calculated specific rates of growth, substrate consumption and product formation were used to diagnose microbial activities during a lactic acid fermentation. The specific rates were calculated from on-line measured cell mass, and substrate and product concentrations. The specific rates were more sensitive indicators of slight changes in fermentation conditions than such monitored data as cell mass or product concentrations.List of Symbols 1/h specific rate of cell growth - 1/h specific rate of substrate consumption - 1/h specific rate of product formation - ^* dimensionless specific rate of cell growth - ^* dimensionless specific rate of substrate consumption - ^* dimensionless specific rate of product formation - _max 1/h maximum specific rate of cell growth - _max 1/h maximum specific rate of substrate consumption - _max 1/h maximum specific rate of product formation - X g/l cell mass concentration - S g/l substrate concentration - S ^* dimensionless substrate concentration - S ₀ g/l initial substrate concentration - P g/l product concentration     

Nucleotide sequence of cytoplasmic 5S ribosomal RNA fromEuglena gracilis

Summary The nucleotide sequence of cytoplasmic 5S ribosomal RNA fromEuglena gracilis has been determined to be: G- _A ^C -G-U-A-C-G-G-C-C-A-U-A-C-U-A-C-C-G-G-G-A-A-U-A-C-A-C-C-U-G-A-A-C-C-C-G--U-C-G-A-U-U-U-C-A-G-A-A-G-U-U-A-A-G-C-C-G-G-G-U-U-A-G-G-C-C-C-A-G-U-U-A-G-U-A-C-U-G-A-G-U-G-G-G-C-G-A-C-C-A-C-U-U-G-G-G-A-A-C-A-C-U-G-G-G-U-G-C-U-G-U-A-C-G-C-U-Up. This RNA is 119 nucleotides long and the sequence of a probable tRNA-binding site is GAUU (position 41–44 from the 5-terminus), which is the same as that of a trypanosoma species,Crithidia fasci-culata. TheEuglena 5S rRNA has a pseudouridine residue at position 38 and 3-terminus is phosphorylated. The 5S rRNA sequence ofEuglena resembles those of several other protozoa and higher animals rather than plants.On leave from Department of Zoology, Hiroshima University, Hiroshima, Japan     

Immunocytochemical markers revealing retinal and pineal but not hypothalamic photoreceptor systems in the Japanese quail

Summary The retinal proteins opsin,-transducin, S-antigen and interstitial retinol-binding protein (IRBP) are essential for the processes of vision. By use of immunocyto-chemistry we have employed antibodies directed against these photoreceptor proteins in an attempt to identify the photoreceptor systems (retina, pineal and deep brain) of the Japanese quail. Opsin immunostaining was identified within many outer (basal portion) and inner segments of retinal photoreceptor cells and limited numbers of photoreceptor perikarya. Opsin immunostaining was also demonstrated in limited numbers of pinealocytes with all parts of these cells being immunoreactive. These results differ from previous observations. In contrast to the results obtained with the antibody against opsin, S-antigen and-transducin immunostaining was seen throughout the entire outer segments and many photoreceptor perikarya of the retina. In the pineal organ immunostaining was seen in numerous pinealocytes in all follicles. These results conform to previous findings in birds. In addition, IRBP has been demonstrated for the first time in the avian retina and pineal organ. These findings underline the structural and functional similarities between the retina and pineal organ and provide additional support for a photoreceptive role of the avian pineal. No specific staining was detected in any other region of the brain in the Japanese quail; the hypothalamic photoreceptors of birds remain unidentified.     

Regular and chaotic behaviour of cardiac cells stimulated at frequencies between 2 and 20 Hz

We measured the non-linear dynamics of cardiac action potentials by varying the stimulation frequency from 2 to 20 Hz and obtained the following results: (i) When the fast Na⁺ current initiated the action potentials (fast action potentials) periodicity was maintained, i.e. the pattern of action potentials repeated after a finite number of stimuli. Chaotic sequences were absent. The transition from one sequence to the next occurred as a devil's staircase. (ii) When, however, the slow Ca²⁺ current initiated the action potentials (slow action potentials), we observed chaos, i.e. fully irregular behaviour, as well as bifurcations. (iii) Our results confirm the supposition that the global dynamics of cardiac cells can be well described by simple one-dimensional maps which predict these two kinds of behaviour.     

Characterization of the MHC class II region in cattle. The number of DQ genes varies between haplotypes

The organization of the major histocompatibility complex (MHC) class II region in cattle was investigated by Southern blot analysis using human probes corresponding to DO, DP, DQ, and DR genes. Exon-specific probes were also employed to facilitate the assessment of the number of different bovine class II genes. The results indicated the presence of single DO and DR genes, at least three DR genes, while the number of DQ genes was found to vary between MHC haplotypes. Four DQ haplotypes, DQ ^{1 1} to DQ ^{2 4}, possessed a single DQ and a single DQ gene whereas both these genes were duplicated in eight other haplotypes, DQ ^{3 5} to DQ ^{9 12}. No firm evidence for the presence of bovine DP genes was obtained. The same human probes were also used to investigate the genetic polymorphism of bovine class II genes. DQ DQ , DR DR , and DO restriction fragment length polymorphisms (RFLPs) were resolved and in particular the DQ restriction fragment patterns were highly polymorphic. Comparison of the present result with the current knowledge of the class II region in other mammalian species suggested that the DO, DP, DQ, DR, and DZ subdivision of the class II region was established already in the ancestor of mammals. The DP genes appear to be the least conserved class II genes among mammalian species and may have been lost in cattle. The degree of polymorphism of different class II genes, as revealed by RFLP analyses, shows striking similarities between species.     

Temperature dependencies and apparent activation energies of stomatal opening and closing

Summary Stomatal opening movements in response to illumination, and stomatal closure following darkening were studied in leaf sections of Zea mays, using air-flow porometers. Stomatal opening is characterized by a phase of linear increase of air flow through the leaf (slope = opening velocity); stomatal closure follows a relaxation curve from which a time constant (closing coefficient) can be derived.Apparent energies of activation, , were computed for the opening velocity and for the closing coefficient from stomatal movements recorded at tissue temperatures between 5° and 50°. It was assumed that the closing coefficient can be used as a measure of the closing force, and that the opening force has to exceed the closing force in order to bring about stomatal opening. is about 7 kcal mole^-1 for the closing coefficient and between 12 and 18 kcal mole^-1 for the opening force. Thus, during stomatal opening, metabolism must provide energy to build up a pressure difference between guard cells and the surrounding tissue.The process controlling the velocity of closure is essentially a passive loss of water (and solutes?) from the guard cells. The of 7 kcal mole^-1 found for the closing coefficient is, however, higher than that for the viscosity of water or for the coefficient of self diffusion of water. It is, therefore, concluded either that water interacts with the cell structures which it has to permeate during stomatal closure, or that the rate of salt loss from guard cells controls the velocity of stomatal closure.The closing force decreases when leaf temperature rises above 35° or falls below 15°. Therefore, stomata of maize open relatively faster and wider above 35° and below 15°, and the 's of the opening velocity appear to be very large above 35° (up to 50 kcal mole^-1) while they have a negative sign below 15°.Research supported by the U.SS. Atomic Energy Commission under contract AT (11-1) 1338. I thank Miss Freia Schulz-Baldes for interested technical assistance.     

Cell recycling studies for α-amylase production by Bacillus amyloliquefaciens

Production of -amylase by a strain of Bacillus amyloliquefaciens was investigated in a cell recycle bioreactor incorporating a membrane filtration module for cell separation. Experimental fermentation studies with the B. amyloliquefaciens strain WA-4 clearly showed that incorporating cell recycling increased -amylase yield and volumetric productivity as compared to conventional continuous fermentation. The effect of operating conditions on -amylase production was difficult to demonstrate experimentally due to the problems of keeping the permeate and bleed rates constant over an extended period of time. Computer simulations were therefore undertaken to support the experimental data, as well as to elucidate the dynamics of -amylase production in the cell recycle bioreactor as compared to conventional chemostat and batch fermentations. Taken together, the simulations and experiments clearly showed that low bleed rate (high recycling ratio) various a high level of -amylase activity. The simulated fermentations revealed that this was especially pronounced at high recycling ratios. Volumetric productivity was maximum at a dilution rate of around 0.4 h^–1 and a high recycling ratio. The latter had to exceed 0.75 before volumetric productivity was significantly greater than with conventional chemostat fermentation.List of Symbols a proportionality constant relating the specific growth rate to the logarithm of G (h) - a ₁ reaction order with respect to starch concentration - a ₂ reaction order with respect to glucose concentration - B bleed rate (h^–1) - C starch concentration (g/l) - C ₀ starch concentration in the feed (g/l) - D dilution rate (h^–1) - D _E volumetric productivity (KNU/(mlh)) - e intracellular -amylase concentration (g/g cell mass) - E extracellular -amylase concentration (KNU/ml) - F volumetric flow rate (l/h) - G average number of genome equivalents of DNA per cell - k _l intracellular equilibrium constant - k ₂ intracellular equilibrium constant - k _s Monod saturation constant (g/l) - k ₃ excretion rate constant (h^–1) - k _d first order decay constant (h^–1) - k _gl rate constant for glucose production - k _st rate constant for starch hydrolysis - k _t1 proportionality constant for -amylase production (gmRNA/g substrate) - k ₁ translation constant (g/(g mRNAh)) - KNU kilo Novo unit - m maintenance coefficient (g substrate/(g cell massh)) - n number of binding sites for the co-repressor on the cytoplasmic repressor - Q repression function K₁/K₂Q1.0 - R ratio of recycling - R _s rate of glucose production (g/lh) - r _c rate of starch hydrolysis (g/(lh)) - R _eX retention by the filter of the compounds X: starch or -amylase - r intracellular -amylase mRNA concentration (g/g cell mass) - r _C volumetric productivity of starch (g/lh) - r _E volumetric productivity of intracellular -amylase (KNU/(g cell massh)) - r _r volumetric productivity of intracellular mRNA (g/(g cell massh)) - r _e volumetric productivity of extracellular -amylase (KNU/(mlh)) - r _s volumetric productivity of glucose (g/(lh)) - r _X volumetric productivity of cell mass (g/(lh)) - S ₀ free reducing sugar concentration in the feed (g/l) - S extracellular concentration of reducing sugar (g/1) - t time (h) - V volume (l) - X cell mass concentration (g/l) - Y yield coefficient (g cell mass/g substrate) - Y _E/S yield coefficient (KNU -amylase/g substrate) - Y _E total amount of -amylase produced (KNU) - substrate uptake (g substrate/(g cell massh)) - specific growth rate of cell mass (h^–1) - _d specific death rate of cells (h^–1) - _m maximum specific growth rate of cell mass (h^–1) This study was supported by Bioprocess Engineering Programme of the Nordic Industrial Foundation and the Center for Process Biotechnology, the Technical University of Denmark.     

The identification of a DNA polymorphism of the α fibrinogen gene,and the regional assignment of the human fibrinogen genes to 4q26-qter

Summary We have identified a common restriction fragment length polymorphism of the fibrinogen gene with the enzyme TaqI. This polymorphism is probably due to a single base change that creates or destroys a TaqI recognition site about 1000 basepairs from the 3 end of the fibrinogen géne. The frequency of the rare allele in 83 unrelated healthy individuals is 0.33. We have used in situ hybridisation of the fibrinogen cDNA to localise the gene on chromosome 4q29–31. We have confirmed this regional localisation by restriction fragment detection in a human x Chinese hamster somatic cell hybrid which contains a translocated human chromosome 4 with a breakpoint at 4q26. The , , and fibrinogen genes are all present on human chromosome 4q26-qter.     

Genetic organization of the E. coli chromosome around the structural gene for initiation factor IF3 (infC).

Summary A set of transducing phages carrying varying lengths of the E. coli chromosome around the structural gene for initiation factor IF3 (infC) was derived from p2 which is known to cary, besides infC, the structural genes for the subunit of phenylalanyl-tRNA synthetase (pheS), the subunit of phenylalanyl-tRNA synthetase (phetT) and the structural gene for threonyl-tRNA synthetase (thrS). The E. coli coding content of these derived phages was analysed by genetic complementation of a set of mutants and by SDS-polyacrylamide gel analysis of the proteins synthesized in UV irradiated cells infected with these phages. The segregation pattern of the different genes among these derived phages indicates that the order of the genes is pheT-pheS-P12-(infC, thrS) where infC is probably between P12 and thrS. P12 is the structural gene of a 12,000 molecular weight unidentified protein.Abbreviations PRS (EC 6.1.1.20) phenylalanyl-tRNA synthetase - TRS (EC 6.1.1.3) threonyl-tRNA synthetase - IF3 Initiation factor IF3 - SDS Sodium dodecyl sulfate - PP^R pyrophosphate resistant - PP^S pyrophosphate sensitive     

Evolutionary temperature adaptation of fish sarcoplasmic reticulum

Summary Sarcoplasmic reticulum has been isolated from the white muscle of 15 species of teleost fish adapted to diverse thermal environments. Evidence has been obtained that the Ca²⁺-dependent ATPase of fish sarcoplasmic reticulum has undergone evolutionary modification for function at different temperatures. Compared with tropical fish, cold adapted species have higher rates of Ca²⁺ transport and Ca²⁺-ATPase activities at low temperatures. Most species have linear Arrhenius plots over the temperature range 0–30°C. Activation enthalpies (H ) of the ATPase ranged from 53–190 kJ mol^–1 and were positively correlated with environment temperature. Activation entropy (S ) varied from negative values in cold adapted species to positive values in tropical fish.In contrast to the Ca²⁺-ATPase, the basal ATPase of fish sarcoplasmic reticulum showed no relationship between either ATPase activity or thermodynamic activation parameters and environmental temperature.Only the Ca²⁺-dependent ATPase is coupled to Ca²⁺ transport. The percentage of total ATPase activity which is Ca²⁺ activated is higher at low temperatures in cold than in warm adapted species. For example, ratios of Ca²⁺-dependent/total ATPase at 2°C varied from 80–98% in Arctic, Antarctic and North Sea species to only 2–50% in various tropical fish. Above 20°C, similar ratios in the range 80–98% were obtained for all species. The nature of the basal ATPase and mechanisms of temperature adaptation of fish sarcoplasmic reticulum are discussed.Abbreviations ET environmental temperature - EGTA ethylene glycol-bis (-aminolethyl ether)-N, N-tetraacetic acid - HEPES N-2-hydroxylpiperazine-N-2-ethanesulfonic acid - SR sarcoplasmic reticulum     

Effect of culture medium ions on chromate reduction by resting cells of Agrobacterium radiobacter

The influence of some ions in pre-growth culture medium on chromate reduction by resting cells of Agrobacterium radiobacter strain EPS-916 was investigated. The reduction was dependent on the Fe²⁺ content of the culture medium: the higher the iron content, the lower the reduction rate. The cells showed maximum chromate reduction when pre-grown in the presence of 0.243 m Mg²⁺, 20 m Ca²⁺ and 3.6 m Mn²⁺. Chromate reduction was not affected by the addition of MgCl₂, CdCl₂, ZnCl₂, MnCl₂, Na₂SO₄ (1000 m), and Na₂MoO₄ (100 m) to the activity assays. However, activity was inhibited by the presence of Na₂SO₄ (10 mm), Na₂MoO₄ (200 m) and ferric citrate.     

Induction of c-mutations in extracellular phage lambda by gamma-rays

Summary Mutagenic action of ⁶⁰Co -rays on extracellular phages red ⁺ and red1 13 after irradiation in 4% nutrient broth in the absence or in the presence of 0.1 M cysteamine or in dried samples was studied. The yield of c mutations was almost independent of the repair genotype of the host cells (uvrA6, polA1, recA13, lexA102, uvrE502, uvrD3 or xthA9), of the phage Red function and of the conditions of -irradiation and was 1·10^-12 per base pair and 1 rad. When the SOS-repair system of the host cells was induced by moderate UV irradiation, the yield of c-mutations was drastically enhanced in phage irradiated in broth, but not in phage irradiated in the dried state. These data allow us to suppose that the direct action of -rays induces, in phage DNA, premutational lesions that are fixed into mutations by replication. On the other hand after -irradiation in broth, when indirect radiation effects are only partially suppressed, about 85% of premutational lesions are converted into mutations by means of the inducible, errorprone SOS-repair system.     

The Synthesis and Antiviral Activity of Glycyrrhizic Acid Conjugates with α-D-Glucosamine and Some Glycosylamines

Glycyrrhizic acid and its 30-methyl ester were conjugated with 2-amino-1,3,4,6-tetra-O-acetyl-2-deoxy--D-glucopyranose, 2,3,4,6-tetra-O-acetyl--D-glucopyranosyl amine, 2,3,4-tri-O-acetyl--L-arabinopyranosyl amine, 2-acetamido-2-deoxy--D-glucopyranosyl amine, and -D-galactopyranosyl amine using N,N-dicyclohexylcarbodiimide and its mixtures with N-hydroxybenzotriazole. Structures of the conjugates were confirmed by IR, UV, ¹H, and ¹³C NMR spectroscopy. The glycoconjugate with the residues of 2-acetamido-2-deoxy--D-glucopyranosyl amine in the carbohydrate part of its molecule exhibited antiviral activity (ID₅₀ 4 g/ml) toward the herpes simplex type 1 virus (HSV-1) in the VERO cell culture. Two compounds demonstrated anti-HIV-1 activity (50–70% inhibition of p24) in a culture of MT-4 cells at concentrations of 0.5–20 g/ml.     

Effect of Naloxone on the Activity of Cl–-Activated Mg2+-ATPase from Plasma Membrane Fraction of Bream Brain (Abramis brama L.) in the Presence of GABAa-ergic Substances

We studied the effect of naloxone—an antagonist of the opioid receptors—on sensitivity of Cl^–-activated Mg²⁺-ATPase from the plasma membrane fraction of bream brain (Abramis brama L.) to GABA_a-ergic substances. Preincubation of the plasma membranes with 1–100 M naloxone increased the basal Mg²⁺-ATPase activity and suppressed its activation by chloride ions. The same effects were observed in the presence of the agonists of GABA_a/benzodiazepine receptors: 0.1–100 M GABA, 1–500 M pentobarbital, and 0.1–100 M phenazepam. Naloxone (10 M) inhibited activation of the basal Mg²⁺-ATPase by the studied ligands and restored the enzyme sensitivity to Cl^–. However, the effect of naloxone was not observed in the presence of high concentrations of pentobarbital (500 M) and phenazepam (100 M). The obtained data show that naloxone modulates the activity of Cl^–-activated Mg²⁺-ATPase from the plasma membranes of bream brain and antagonizes the GABA_a receptor ligands.     

Freeze-fracture studies on the cockroach hemocyte membrane complex: Symmetric and asymmetric membrane particle distributions

Summary Freeze-fracture studies were conducted on the membranes of normal cockroach hemocytes. The plasmalemma is asymmetric with the A fracture face containing 80–100 Å membrane intercalated particles at a concentration of 2500/². The B fracture face contains 120–150 Å particles with a relatively low density (800/²). The nuclear envelope displays an asymmetry with the A fracture face containing 1500 particles/² and the B face containing 300/ ². No significant particle size differences were observed in nuclear envelope fracture faces. Two types of symmetric membranes were also found in these cells. Both A and B fracture faces of the membrane surrounding the numerous cytoplasmic inclusion bodies contain particle sizes and concentrations similar to the B face of the plasmalemma. A second type of symmetry was observed in cells apparently engaged in exocytosis. Vesicles (0.1 D) from this process were completely particle free on both fracture faces. Such particle free vesicles could be found in the cytoplasm, attached to the plasmalemma, or completely separated from the cell.Supported by a Pharmaceutical Manufacturers Association Foundation Fellowship.The author wishes to thank Ms. Annalena K. Charla for assistance in plate preparation, Dr. Julius Schultz and the Papanicolaou Cancer Research Institute for use of the freeze-etch device, and Dr. David Smith for the electron microscope facilities.     

Factors influencing shoot regeneration from cotyledonary explants ofCucumis melo

Cotyledonary explants of 4-day-oldCucumis melo cv. Hale's Best Jumbo in vitro seedlings showed maximum initiation of shoot buds when cultured onto a revised Murashige & Skoog medium supplemented with 5 M indole-3-acetic acid and 5 M benzylaminopurine and cultured at 25–29°C under low light intensity (5–30 mol m^-2 s^-1). Subculture of the shoot buds onto the same medium without auxin and supplemented with 3 M benzylaminopurine caused the development of shoots from 30% of the buds. The presence of abscisic acid significantly increased the number of explants producing shoot buds. Bud initiation was affected by genotype, seedling age, light intensity, and temperature. Addition of gibberellic acid, thidiazuron or silver nitrate to regeneration medium did not improve either bud initiation or shoot regeneration.     

Activation of protein kinase C alpha is required for TPA-triggered ERK (MAPK) signaling and growth inhibition of human hepatoma cell HepG2

The signaling mechanisms for most of the antiproliferative processes are not fully understood. We have demonstrated that ERK(MAPK) signaling was involved in the induction of both p15^INK4band p16^INK4a CDK inhibitors and growth inhibition of hepatoma cell HepG2 triggered by the tumor promoter tetradecanoyl phorbol acetate (TPA). In this study, the upstream signal mechanism for TPA-induced ERK(MAPK) activation was investigated. In HepG2 cells only one of the cPKC isozymes, PKC, but not cPKCII, nPKC or aPKC was activated by TPA as demonstrated by its membrane translocation within 10–30 min and down-regulation at 24 h after TPA treatment. Pretreatment of 0.2–2.0 M Bisindolylmaleimides, an inhibitor of PKC, attenuated the TPA-induced phosphorylation of ERK, gene expressions of p15^INK4band p16^INK4a, and growth inhibition of HepG2 cell in a dose-dependent manner. Consistently, transfection of HepG2 with 1.0–3.0 M antisense (AS) PKC, but not (AS) PKCII, or nPKC oligonucleotides (ODN), for 36 h prior to TPA treatment also prevented the TPA-induced molecular and cellular effects described above. Taken together, we concluded that PKC is specifically required for TPA-induced ERK(MAPK) signaling to trigger gene expressions of p15^INK4band p16^INK4a leading to HepG2 growth inhibition.