Glucose and lactate utilization by the amygdala of male and female rats

Gonadal hormones appear to modulate brain energy metabolism, and morphological and functional sexual differences are found in the amygdaloid complex (AC) of rats. Our aim was to study the CO₂ production and lipid synthesis, measured by the rate of L-[U-¹⁴C]lactate or D-[U-¹⁴C]glucose utilization (in pmol.hr^–1.mg^–1), by AC slices in vitro of male and female rats. Lactate was more used than glucose as energy substrate (p < 0.01) but no sex-related difference was observed in glucose or lactate oxidation to CO₂ (p > 0.05) or on lipid synthesis obtained from both substrates (p > 0.05). In addition, there was no effect of the estrous cycle on lactate oxidation to CO₂ by the AC of females (p > 0.05). Based on the present data, it appears that the endogenous normal levels of gonadal hormones are not able to promote sex-related differences in the in vitro glucose or lactate utilization by the AC of rats.     

Cloning and expression of chondroitinase AC from Bacteroides stercoris HJ-15

Enzymes that degrade glycosaminoglycans (GAGs) can help reveal the biological roles, structure, and mechanisms of GAGs. We cloned chondroitinase AC, which can degrade chondroitin sulfates A and C, from the genomic library of Bacteroides stercoris HJ-15 isolated from human intestine. The probe (1.4 kb) for the chondroitinase AC gene was prepared from the PCR product of the primers produced using two internal amino acid sequences of chondroitinase AC purified from B. stercoris HJ-15. Using this probe, a chondroitinase AC-positive, 4 kb DNA fragment was selected from pKF3 vector gene libraries containing 2.5–4.5 kb DNA fragments digested with HindIII. The amino acid sequence of the cloned chondroitinase AC showed 41% homology to that of Flavobacterium heparinum. The cloned chondroitinase AC gene was expressed under the T7 promoter of the expression vector, pET-26b(+), in Escherichia coli BL21(DE3) and purified using His bind column chromatography. The expressed chondroitinase AC potently degraded chondroitin sulfates A and C.     

In vitro study of alginate–chitosan microcapsules: an alternative to liver cell transplants for the treatment of liver failure

The application of alginate–chitosan (AC) microcapsules to liver cell transplantation has not been previously investigated. In the current in vitro study, we have investigated the potential of AC microcapsules for the encapsulation of liver cells and show that the AC membrane supports the survival, proliferation and protein secretion by entrapped hepatocytes. The AC membrane provides cell immuno-isolation and has the potential for cell cryopreservation. The AC microcapsule has several advantages compared to more widely used alginate–poly-L-lysine (APA) microcapsules for the application of cell therapy.     

Isolation of <Emphasis Type="BoldItalic">Serratia marcescens</Emphasis> as a chondroitinase-producing bacterium and purification of a novel chondroitinase AC

A strain of Serratia marcescens that produced chondroitinase was isolated from soil. It produced a novel chondroitinase AC, which was purified to homogeneity. The enzyme was composed of two identical subunits of 35 kDa as revealed by SDS-PAGE and gel filtration. The isoelectric point for the chondroitinase AC was 7.19. Its optimal activity was at pH 7.5 and 40 °C. The purified enzyme was active on chondroitin sulfates A and C and hyaluronic acid, but was not with chondroitin sulfate B (dermatan sulfate), heparin or heparan sulfate. The apparent K_m and V_max of the chondroitinase AC for chondroitin sulfate A were 0.4 mg ml^–1 and 85 mmol min^–1 mg^–1, respectively, and for chondroitin sulfate C, 0.5 mg ml^–1 and 103 mmol min^–1 mg^–1, respectively.     

Biodegradation of naphthalene-2-sulfonic acid present in tannery wastewater by bacterial isolates <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. 2AC and <Emphasis Type="Italic">Comamonas</Emphasis> sp. 4BC

Two bacterial strains, 2AC and 4BC, both capable of utilizing naphthalene-2-sulfonic acid (2-NSA) as a sole source of carbon, were isolated from activated sludges previously exposed to tannery wastewater. Enrichments were carried out in mineral salt medium (MSM) with 2-NSA as the sole carbon source. 16S rDNA sequencing analysis indicated that 2AC is an Arthrobacter sp. and 4BC is a Comamonas sp. Within 33 h, both isolates degraded 100% of 2-NSA in MSM and also 2-NSA in non-sterile tannery wastewater. The yield coefficient was 0.33 g biomass dry weight per gram of 2-NSA. A conceptual model, which describes the aerobic transformation of organic matter, was used for interpreting the biodegradation kinetics of 2-NSA. The half-lives for 2-NSA, at initial concentrations of 100 and 500 mg/l in MSM, ranged from 20 h (2AC) to 26 h (4BC) with lag-phases of 8 h (2AC) and 12 h (4BC). The carbon balance indicates that 75–90% of the initial TOC (total organic carbon) was mineralized, 5–20% remained as DOC (dissolved organic carbon) and 3–10% was biomass carbon. The principal metabolite of 2-NSA biodegradation (in both MSM and tannery wastewater) produced by Comamonas sp. 4BC had a MW of 174 and accounted for the residual DOC (7.0–19.0% of the initial TOC and 66% of the remaining TOC). Three to ten percent of the initial TOC (33% of the remaining TOC) was associated with biomass. The metabolite was not detected when Arthrobacter sp. 2AC was used, and a lower residual DOC and biomass carbon were recorded. This suggests that the two strains may use different catabolic pathways for 2-NSA degradation. The rapid biodegradation of 2-NSA (100 mg/l) added to non-sterile tannery wastewater (total 2-NSA, 105 mg/l) when inoculated with eitherArthrobacter 2AC or Comamonas 4BC showed that both strains were able to compete with the indigenous microorganisms and degrade 2-NSA even in the presence of alternate carbon sources (DOC in tannery wastewater = 91 mg/l). The results provide information useful for the rational design of bioreactors for tannery wastewater treatment.     

Regulation of Adenylyl Cyclase Signaling System in Cell Cultures of Infusoria Dileptus anser and Tetrahymena pyriformis by Peptides of Insulin Superfamily

Hormone-sensitive adenylyl cyclase signaling system (ACS) provides transduction of a wide spectrum of hormonal signals in cells of the higher eucaryotes. At the same time, ACS in the lower eucaryotes at present is practically not studied. We studied regulatory effects on ACS of the infusoria Dileptus anser and Tetrahymena pyriformis of peptide hormones of the higher eukaryotes—insulin, IGF-1, and relaxin, whose action on ACS of the higher eucaryotes was the subject of our earlier studies. The action of these hormones at concentrations of 10^–10–10^–8 M on the AC activity in infusoria had clearly stimulating character, the dose–effect curves being of a bell-shaped form with a maximum of the stimulating effect of the hormones at concentrations of 10^–9–10^–8 M. the shape of the curves and the value of the stimulating effect of the peptide hormones depended substantially on the level of the AC basal activity in homogenates of infusorian cell cultures. All the hormones (10^–8 M) stimulated GTP-binding activity of G-proteins. It was shown by the example of relaxin that its stimulating effect on GTP-binding in infusorian cells was dose-dependent and increased in the range of hormone concentrations from 10^–10 to 10^–8 M to reach its maximum at concentrations of 10^–8–10^–7 M. In the presence of suramin, an inhibitor of heterotrimeric G-proteins, the stimulating effects of the hormones on the GTP-binding and the AC activity decreased essentially or were absent completely. This indicates that the heterotrimeric G-proteins are ones of components of the signaling cascade that mediates regulatory effects of the hormones of the insulin group on the AC activity in infusorian cell cultures. Based on the obtained data, it is suggested that the basic molecular mechanisms of regulation of ACS by insulin and the related peptides that are similar to those found in the higher vertebrates already begin to be formed as early as at the level of the lower eucaryotes.     

Growth,Stomatal Conductance,Photosynthetic Rate,Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Phosphoenolpyruvate Carboxylase Activities during Rooting and Acclimatisation of Rosa Hybrida Plantlets

The rooting of shoots of micropropagated Rosa hybrida cv. Madame Delbard was conducted on MS medium with 30 kg m^–3 sucrose or on hydroponic medium (containing less mineral salts), under higher photosynthetic photon flux density (PPFD) (100 in comparison with 45 µmol m^–2 s^–1) and flushed by ambient air [AC, 340 µmol(CO₂) mol^–1] or by CO₂-enriched air (EC, 2 500 µmol mol^–1) and lower relative humidity (80–90 % vs. 96–99 %). This cultivation led to plantlets with longer roots and adventitious root formation. Net photosynthetic rate and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) activities, RuBPCO/phosphoenolpyruvate carboxylase activities ratio, and starch accumulation increased under these conditions. After 14 d, plantlets had functional stomata and could be acclimated on open benches without gradual decrease in relative humidity. The percentage of survival was higher when the rooting took place in EC than in AC. However, the advantage acquired during rooting phase by plantlets cultured in liquid medium was not maintained after 4 weeks of acclimatisation.     

Mapping of adenylyl cyclase genes type I,II, III,IV, V,and VI in mouse

The adenylyl cyclases (AC) act as second messengers in regulatory processes in the central nervous system. They might be involved in the pathophysiology of diseases, but their biological function is unknown, except for AC type I, which has been implicated in learning and memory. We previously mapped the gene encoding AC I to human Chromosome (Chr) 7p12. In this study we report the mapping of the adenylyl cyclase genes type I–VI to mouse chromosomes by fluorescence in situ hybridization (FISH): Adcy1 to Chr 11A2, Adcy2 to 13C1, Adcy3 to 12A-B, Adcy4 to 14D3, Adcy5 to 16B5, and Adcy6 to 15F. We also confirmed previously reported mapping results of the corresponding human loci ADCY2, ADCY3, ADCY5, and ADCY6 to human chromosomes and, in addition, determined the chromosomal location of ADCY4 to human Chr 14q11.2. The mapping data confirm known areas of conservation between mouse and human chromosomes.     

Chromatographic Separation and Assignment of Absolute Configuration of Hydroxywarfarin Isomers

The absolute configuration of several hydroxywarfarin isomers was assigned using a comparison of elution order on chiral stationary phases, optical rotation, and circular dichroism (CD) spectra, with confirmation of assignments made by comparison between experimental and calculated CD spectra and selective synthesis of hydroxywarfarin isomers from enantiopure warfarin using human liver microsomes. Chirality 26:95–101, 2014. © 2013 Wiley Periodicals, Inc.     

Somatic embryogenesis in loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis>) and Douglas fir (<Emphasis Type="Italic">Pseudotsuga menziesii</Emphasis>): improving culture initiation and growth with MES pH buffer,biotin, and folic acid

Loblolly pine (Pinus taeda L.) somatic embryogenesis initiation was improved by supplementing the initiation medium with the pH buffer agent 2(n-morpholino)ethanesulphonic acid (MES) at 250 mg l^–1, folic acid at 0.5 mg l¹, and biotin at 0.05 mg l^–1. MES and vitamins increased the percentage of explants with extruded tissue that continued the initiation process to form embryogenic tissue. The increase in initiation was about 12%. Initiation of 12 open-pollinated families averaged 38.5%, which is 16% higher than initiation on medium without these additives. When tested with 18 control-pollinated families, initiation averaged 26.3%. Basal medium contained a combination of modified 1/2 P6 salts, activated carbon (AC) at 50 mg ^–1, Cu and Zn adjusted to compensate for adsorption by AC, 1.5% maltose, 2% myo-inositol, 500 mg l^–1 casamino acids, 450 mg l^–1 glutamine, 2 mg l^–1 NAA, 0.63 mg l^–1 BAP, 0.61 mg l^–1 kinetin, 3.4 mg l^–1 silver nitrate, 10 M 8-Br-cGMP, 0.1 M brassinolide, and 2 g l^–1 Gelrite. Early-stage embryo growth and initiation in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) were also improved in the presence of these additives.     

Revised Absolute Configuration of Sibiricumin A: Substituent Effects in Simplified Model Structures Used for Quantum Mechanical Predictions of Chiroptical Properties

This study discusses the choice of different simplified models used in computations of electronic circular dichroism (ECD) spectra and other chiroptical characteristics used to determine the absolute configuration (AC) of the complex natural product sibiricumin A. Sections of molecules containing one chiral center with one near an aromatic group have large effects on the ECD spectra. Conversely, when the phenyl group is present on a substituent without a nonstereogenic center, removal of this section will have little effect on ECD spectra. However, these nonstereogenic‐center‐containing sections have large effects on calculated optical rotations (OR) values since the OR value is more sensitive to the geometries of sections in a molecule. In this study, the wrong AC of sibiricumin A was reassigned as (7R,8S,1'R,7'R,8'S)‐ 11 . Chirality 28:612–617, 2016. © 2016 Wiley Periodicals, Inc.     

Myosin heavy chain isoforms expression and cyclic AMP concentrations in hypoxia-induced hypertrophied right ventricle in rats

We have previously demonstrated that the relative expression of myosin heavy chain-beta (MHC-β) in both ventricles of rats exposed to long-term hypobaric hypoxia correlated significantly with the relative ventricular mass. In the present study, we investigated whether an increased expression of MHC-β was accompanied by a reduction in cyclic AMP (cAMP) activity in hypoxia-induced hypertrophied right ventricle (RV). We used male Wistar–Kyoto rats born and raised at simulated altitudes (2200 m: H2 group or 4000 m: H4 group) compared to age-matched sea level controls (SC group). There were no significant differences between the groups in basal and forskolin-stimulated adenylyl cyclase (AC) activities. The basal and IBMX-inhibited phosphodiesterase (PDE) activities were slightly higher in both hypoxic groups (p>0.05), except that the H2 group had a higher basal PDE activity than the SC group (p<0.05). The AC/PDE activity ratios were significantly decreased in both hypoxic groups (p<0.05), suggesting that low concentrations of cellular cAMP were maintained in the RV under hypoxic conditions. However, there were no correlations between MHC-β expression and either AC activity, PDE activity, or AC/PDE activity ratio. These results provided evidence against the causal role for cAMP concentration in the expression of MHC-β associated with hypoxia-induced ventricular hypertrophy.     

Streptomycin-induced ribosome engineering complemented with fermentation optimization for enhanced production of 10-membered enediynes tiancimycin-A and tiancimycin-D

Tiancimycins (TNMs) are a group of 10-membered anthraquinone-fused enediynes, newly discovered from Streptomyces sp. CB03234. Among them, TNM-A and TNM-D have exhibited excellent antitumor performances and could be exploited as very promising warheads for the development of anticancer antibody-drug conjugates (ADCs). However, their low titers, especially TNM-D, have severely limited following progress. Therefore, the streptomycin-induced ribosome engineering was adopted in this work for strain improvement of CB03234, and a TNMs high producer S. sp. CB03234-S with the K43N mutation at 30S ribosomal protein S12 was successfully screened out. Subsequent media optimization revealed the essential effects of iodide and copper ion on the production of TNMs, while the substitution of nitrogen source could evidently promote the accumulation of TNM-D, and the ratio of produced TNM-A and TNM-D was responsive to the change of carbon and nitrogen ratio in the medium. Further amelioration of the pH control in scaled up 25 L fermentation increased the average titers of TNM-A and TNM-D up to 13.7 ± 0.3 and 19.2 ± 0.4 mg/L, respectively. The achieved over 45-fold titer improvement of TNM-A, and 109-fold total titer improvement of TNM-A and TNM-D enabled the efficient purification of over 200 mg of each target molecule from 25 L fermentation. Our efforts have demonstrated a practical strategy for titer improvement of anthraquinone-fused enediynes and set up a solid base for the pilot scale production and preclinical studies of TNMs to expedite the future development of anticancer ADC drugs.     

The pharmacodynamic action of the cyclic AMP phosphodiesterase inhibitor rolipram on prolactin producing rat pituitary adenoma (GH4C1) cells

Rolipram (4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone) represents a new class of specific low K_m cAMP phosphodiesterase (PDE) inhibitors. This compound enhances basal, hormone- and forskolin-elicited cAMP accumulation in prolactin (PRL) producing rat pituitary adenoma (GH₄C₁) cells in culture (ED₅₀=5·10^–8 M). This effect is due to a selective inhibition of the low K_m cAMP PDE (type III), since neither basal nor hormone-stimulated adenylate cyclase (AC) nor the Ca²⁺/calmodulin-dependent PDE were affected by rolipram. The drug enhanced vasoactive intestinal polypeptide (VIP)-stimulated PRL-secretion, while thyroliberin (TRH)- and 12-0-tetradecanoyl phorbol-13-acetate (TPA)-elicited PRL egress were slightly reduced indicating a cAMP-mediated reduction of protein kinase C (PK-C) mediated PRL release. Interestingly, inhibition of PRL secretion by somatostatin (SRIH) was completely suppressed suggesting cAMP-mediated inactivation of some GTP-binding protein(s) of the _i family (G _i2 orG _k). Rolipram did not affect phosphoinositide metabolism (i.e. IP₃ accumulation), neither acutely nor after long term administration. Rolipram, like the cAMP PDE inhibitor Ro 20–1724, did not influence AC and PDE I, but dose-dependently inhibited PDE III activity.Long term incubation of GH₄C₁ cells with rolipram in the presence of noradrenaline (NA) exerted a marginal decrease of -receptor number, AC activation and cAMP accumulation, while Ro 20–1724 brought about a marked down-regulation and desensitization of the AC complex.In summary, rolipram selectively interacts with PDE III in rat pituitary adenoma cells in culture and does not result in -adrenoceptor AC downregulation. These features are not shared by the other drugs tested.     

Modeling available 2,4-dichlorophenoxyacetic acid in a tissue culture medium containing activated carbon

Clonal propagation of high-value forest trees by somatic embryogenesis can help meet industry needs for uniform and high quality raw materials. Low embryogenic tissue initiation frequencies for loblolly pine (Pinus taeda L.) pose a limitation in work towards commercialization of this technology. At the time our research began most work on somatic embryo culture initiation in loblolly pine reported success in the range of 1–5%. Activated carbon (AC) has been reported to improve many tissue culture systems including embryogenic tissue initiation in Douglas-fir. To improve initiation frequencies in loblolly pine, the development of an AC-containing system was explored. In order to better understand the availability of 2,4-dichlorophenoxyacetic acid (2,4-D) in initiation medium, we tracked media surface concentrations of free or available 2,4-D. Media containing 1/2 modified P6 salts, 1.5% maltose, 2% myo-inositol, case amino acids, glutamine, vitamins, and 0.4% Gelrite were modified to include 0.625 – 2.5 g l^–1 of activated carbon (Sigma C-9157, acid washed) and 110 –440 mg l^–1 2,4-D. Adsorption and availability of 2,4-D in AC-containing medium was tracked by C¹⁴ labeled 2,4-D present in surface moisture absorbed into filter paper. High correlations were found between–available 2,4-D and time when AC and initial 2,4-D concentrations were held constant,–available 2,4-D and AC concentration when initial added 2,4-D and time were held constant, and–available 2,4-D and initial 2,4-D when AC and time were held constant.All of these relationships were exponential, not linear. Multiple regression models inputting initial 2,4-D added to medium in mg l^–1, activated carbon added to medium in %, and time in days, were able to explain 85–88% of the variability in available 2,4-D. These models can be used to achieve target levels of available 2,4-D by adjustment of initial 2,4-D levels or AC content.     

Short- versus Long-Term Effects of Elevated CO2 on Night-Time Respiration of Needles of Scots Pine (Pinus sylvestris L.)

Dark respiration rate in the night (R _D) was measured in five-year-old Scots pine (Pinus sylvestris L.) trees grown for two years under ambient (AC) and elevated (AC + 400 µmol mol^–1 = EC) CO₂ concentrations in open top chambers. Two needle age classes (i.e., current-year and one-year-old) were measured at AC and EC in both AC- and EC-grown pines. Additionally different chemical characteristics were determined on the needles, such as nitrogen (N), carbon (C), starch, and soluble sugar concentrations as well as specific leaf area. The direct, short-term and indirect, long-term effects of EC on R _D for the two needle age classes were examined. R _D was expressed on a per needle area, needle mass, N, C, and C/N bases. Direct effects were only pronounced in the AC treatment where inhibition of R _D was found at EC in both current- and one-year-old needles. Indirect effects were only significant in one-year-old needles where a decrease was found in the EC grown trees as compared with AC ones when R _D was expressed per unit needle mass, C, or C/N. R _D per unit needle area and needle N were not sensitive to long-term EC, in any needle age class. Long-term EC treatment also influenced the response of the two needle age classes. One-year-old needles from the EC treatment had significantly lower R _D than current-year needles, but no such response was observed in the AC treatment. Our experiment re-emphasised the importance of expressing R _D on different bases for a correct interpretation of the responses to EC. Moreover, we showed that different needle age classes can respond differently to a CO₂ enrichment.     

Effects of activated charcoal, explant size, explant position and sucrose concentration on plant and shoot regeneration of Lilium longiflorum via young stem culture

An efficient system for the in vitro plant and shootregeneration of Lilium longiflorum was developed andaccomplished using transverse thin cell layers (tTCL) of young stems.tTCLs were cut transversely along young stems from which the shoot-tipshad been removed. Sections were measured accurately using a graded gridand were cut in 4 mm × 4 mm × 1 mm cubes, eliminatingepidermal tissue, and were cultured on one-half MS medium containing 8 gl^–1 agar, different sucrose concentrations (10, 20, 30 or 40g l^–1), and with or without 1 mg l^–1 activatedcharcoal (AC). Plants formed on the surface of tTCLs within 60 days onone-half MS medium containing 8 g l^–1 agar and 20 gl^–1 sucrose. Sections of 1 mm taken just below the apicalarea developed buds within 15 days, whereas the sections closer to thebase required about 45 days. Shoot regeneration was enhanced whensucrose concentration was used at 30 or 40 g l^–1 after 60days of culture. No root formation occurred. Both shooting and rootingoccurred when sucrose was used at 20 g l^–1. The plantletswere transferred to soil and grew well under greenhouseconditions.     

Low baseline ribosome-related gene expression and resistance training-induced declines in ribosome-related gene expression are associated with skeletal muscle hypertrophy in young men and women

Ribosomes are essential cellular machinery for protein synthesis. It is hypothesised that ribosome content supports muscle growth and that individuals with more ribosomes have greater increases in muscle size following resistance training (RT). Aerobic conditioning (AC) also elicits distinct physiological adaptations; however, no measures of ribosome content following AC have been conducted. We used ribosome-related gene expression as a proxy measure for ribosome content and hypothesised that AC and RT would increase ribosome-related gene expression. Fourteen young men and women performed 6 weeks of single-legged AC followed by 10 weeks of double-legged RT. Muscle biopsies were taken following AC and following RT in the aerobically conditioned (AC+RT) and unconditioned (RT) legs. No differences in regulatory genes (Ubf, Cyclin D1, Tif-1a and Polr-1b) involved in ribosomal biogenesis or ribosomal RNA (45S, 5.8S, 18S and 28S rRNAs) expression were observed following AC and RT, except for c-Myc (RT > AC+RT) and 5S rRNA (RT < AC+RT at pre-RT) with 18S external transcribed spacer and 5.8S internal transcribed spacer expression decreasing from pre-RT to post-RT in the RT leg only. When divided for change in leg-lean soft tissue mass (ΔLLSTM) following RT, legs with the greatest ΔLLSTM had lower expression in 11/13 measured ribosome-related genes before RT and decreased expression in 9/13 genes following RT. These results indicate that AC and RT did not increase ribosome-related gene expression. Contrary to previous research, the greatest increase in muscle mass was associated with lower changes in ribosome-related gene expression over the course of the 10-week training programme. This may point to the importance of translational efficiency rather than translational capacity (i.e. ribosome content) in mediating long-term exercise-induced adaptations in skeletal muscle.     

Adenylyl Cyclase Signaling Mechanism of Relaxin Action

In connection with our discovery of the adenylyl cyclase signaling mechanism (ACSM) of action of some peptides belonging to the insulin superfamily, a possibility of its involvement in action of another insulin superfamily peptide, relaxin, was studied. It was shown for the first time that human relaxin-2 (10^–12–10^–8 M) activated adenylyl cyclase (AC) in a dose-dependent manner. The maximal peptide effect was revealed at a concentration of 10^–8 M. Under condition of the hormonal action the basal enzyme activity increased by +310% in human myometrium, by +117%, in rat skeletal muscles, and by +49%, in foot smooth muscles of the bivalve mollusc Anodonta cygnea. Insulin and mammalian insulin-like growth factor-I (IGF-I) also produced the AC activating effect in these muscles. The order of efficiency of these peptides, based on their ability to induce the maximal AC stimulating effect, was as follows: relaxin > IGF-I > insulin (human myometrium); IGF-I > relaxin > insulin (rat skeletal muscle); insulin-like peptide of Anodonta (ILPA) > IGF-I > insulin > relaxin (molluscan muscle). The relaxin activating effect on AC was potentiated by a guanine nucleotide, the non-hydrolyzed analog of GTP, guanylylimidodiphosphate (Gpp[NH]p), which indicates participation of G_s-protein in realization of this effect. This effect was inhibited by a tyrosine kinase selective blocker, tyrphostin 47, and a phosphatidylinositol-3-kinase (PI-3-K) selective blocker, wortmannin. Thus, for the first time, participation of ACSM in the relaxin action has been established. This mechanism, as suggested at the present time state of its study, includes the following signal pathway: receptor-tyrosine kinase PI-3-K G_s-protein AC.     

Vector-free cloning of a bacterial endo-1,4-β-glucanase in Lactobacillus plantarum and its effect on the acidifying activity in silage: Use of recombinant cellulolytic Lactobacillus plantarum as silage inoculant

In this research, the advantage of use of cellulolytic recombinant Lactobacillus plantarum as microbial inoculants for alfalfa silage fermentation was evaluated. To such purpose, two L. plantarum strains, one (L. plantarum Lp80) currently commercialised and the other (L. plantarum B41) suitable as silage microbial additive, were genetically modified by integration of celA gene, encoding an alkaline endo-1,4--glucanase from Bacillus sp., in the chromosome, by means of a vector-free cloning technique. The heterologous gene was cloned in two fashions: preceded by two promoters (AC1 modification) or in translational coupling with a partial upstream ORF (AC2 modification). Therefore two different genetically modified organisms (GMOs) per each wild-type (WT), producing 43–59 U/l cellulase in 16 h, were examined. Thirty-five micro-ensiling experiments were carried out by inoculating the WT or the derived GMOs. L. plantarum B41AC1 cellulolytic clone exhibited significantly increased acidification capacity in silage samples incubated at 37°C. No advantage of use was evident for the other GMOs.