Effects of oxygen partial pressure and combined nitrogen on N2-fixation (C2H2) associated withZea mays and other gramineous species

Summary The objectives of this investigation were to determine the effects of oxygen partial pressure (pO₂) and combined nitrogen (NH ₄ ⁺ ) on rates of acetylene reduction (AR) associated with roots of intact corn, sorghum, and pearl millet plants. Soil-grown plants were carefully removed from soil and incubated hydroponically with the root system enclosed in a plastic cylinder; the tops were left exposed to ambient conditions. Oxygen concentrations around the root systems were controlled by sparging the nutrient solution with known quantities of O₂ in N₂. Ammonium nitrogen was added to the nutrient solution following establishment of AR rates to determine its effect on rates of N₂-fixation (AR). Substantial AR rates (0.1–1.5 mol C₂H₄ g dry wt^–1 h^–1) were associated with roots exposed to 0–2% O₂ (v/v) (0.0–2.02 kPa) in N₂ following at 12–24 h period of exposure to the reduced oxygen tension. Root systems exposed to air failed to demonstrate AR while those exposed to 100% N₂ showed lower activity than those at reduced pO₂ values. Addition of NH ₄ ⁺ (10–20 g N ml^–1 of nutrient solution) reduced AR by 75–90% within 24 h after addition. Oxygen uptake by roots exposed to low pO₂ was substantially reduced.     

Influence of cadmium on certain biological activities in a cadmium-tolerant fungi

Aspergillus carbonarius and a strain ofPenicillium are able to grow on Harrold's agar media amended with different concentrations of cadmium chloride up to 2.5% (w/v). Considerable quantities of cadmium were absorbed by both fungi.A. carbonarius absorbed more cadmium than thePenicillium sp. did, under the same culturing conditions. In the presence of cadmium, the determined cellular contents of proteins, lipids, and carbohydrates were extraordinary high, whereas the activities of certain enzymes, lipases, amylases, and proteases were inhibited. The fungal rate of growth and sporulations were mostly suppressed. Conidiations were inhibited at lowest concentrations. At 1% Cd Cl₂,A. carbonarius produced malformed conidiophores, whereas thePenicillium sp. was less affected. At higher concentrations conidiophores production were entirely suppressed and several hyphal swellings were produced.     

Encapsulation of Lipophilic Bioactive Molecules by Microchannel Emulsification

Currently, much effort is being invested in novel formulations of bioactive molecules, such as emulsions, for pharmaceutical, food, and cosmetic applications. Therefore, methods to produce emulsions with controlled-size droplets of uniform size distribution have been developed. On this concern, a microfluidic device called the microchannel (MC) was used in this work for emulsification. This is a novel method for producing monodispersed emulsion droplets with very narrow droplet size distribution and low energy input, due to the spontaneous droplet generation basically driven by the interfacial tension, unlike other conventional emulsification processes. This technology provides the formulation of oil-in-water (O/W) emulsions containing lipophilic active molecules with increased bioavailability, which may be readily absorbed by the human body. MC emulsification enables the preparation of highly monodispersed O/W emulsions, which may be applied as enhancer on active molecules delivery systems, as well as in foodstuff. In this study, formulations of O/W emulsions loaded with bioactive molecules, such as β-carotene and γ-oryzanol, were prepared by the MC emulsification process. Refined soybean oil containing the dissolved lipophilic molecule and either sugar ester or gelatin solution (1 wt.%) were used as the dispersed and continuous phases, respectively. The emulsification process conducted using the asymmetric straight-through MC plate enabled the production of monodispersed O/W emulsions, resulting in β-carotene-loaded O/W emulsions with average droplet size (d _av) of 27.6 μm and coefficient of variation (CV) of 2.3% and γ-oryzanol-loaded droplets with d _av of 28.8 μm and CV of 3.8%. The highly monodisperse β-carotene-loaded droplets were physically stable throughout the storage period observed, resulting in droplets with d _av 28.2 μm and CV of 2.9% after 4 months storage in darkness at 5 °C. Single micrometer-sized monodisperse emulsions loaded with β-carotene were successfully formulated using the grooved MC emulsification, resulting in droplets with d _av of 9.1 μm and CV of 6.2%. This work was funded by The Ministry of Agriculture, Forestry and Fisheries of Japan, through the Food Nanotechnology Project, and the Japan Society for the Promotion of Science.     

Degradation of different hydrocarbons and production of biosurfactant byPseudomonas aeruginosa isolated from coastal waters

Summary A bacterium isolated from an oil spillage sample and identified asPseudomonas aeruginosa degraded hexadecane and heptadecane by 47% and 58% with 9% and 12% of total carbon from the respective substrates being liberated as CO₂. With octadecane and nonadecane as substrates, 73% and 60% were biodegraded while 27% and 25% of total carbon was evolved as CO², respectively. Production of biosurfactant by this bacterium was studied using hexadecane 5% (v/v) as substrate. The surface tension of spent culture medium, as well as the supernatant, was 30 mN/m compared to 71 mN/m for water. When the supernatant was mixed with hexadecane (15, v/v) a stable emulsion was formed which deteriorated only by 10% after one month.

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Dégradation de différents hydrocarbures et production de biosurfactants parPseudomonas aeruginosa isolé d'eaux côtières

Résumé Une bactérie, isolée d'un échantillon d'hydrocarbures épandus, et identifiée commePseudomonas aeruginosa, dégrade l'hexaet l'heptadécane à raison respective de 47 et de 58% du carbone total avec 9 et 12% des substrats respectifs libérés sous la forme de CO₂. Avec l'octa- et le nonadécane comme substrats, la biodégradation atteint respectivement 73 et 60% avec 27 et 25% du carbone total transformé en CO₂. On a étudié la production de biosurfactant par cette bactérie en utilisant l'hexadécane à 5% (v/v) comme substrat. La tension superficielle de la liqueur mixte, comme celle de son surnageant atteint 30 mN/m par comparaison à 71 mN/m pour l'eau. Lorsque le surnageant est mélangé à l'hexadécane (15, v/v), on forme une émulsion stable qui ne se détériore que de 10% au bout d'un mois.

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This work was carried out at the Biotechnology Laboratories of the Nuclear Institute for Agriculture & Biology (NIAB), Faisalabad.     

Isolation,Identification and Screening of Manganese Solubilizing Fungi From Low-Grade Manganese Ore Deposits

The present investigation reports the isolation, molecular identification and screening of manganese (Mn) solubilizing fungal strains from low-grade Mn mine tailings. Six morphologically distinct Mn solubilizing fungal strains were isolated on MnO₂-supplemented agar plates with Mn concentration of 0.1% (w/v). The biochemical characterization of the isolated fungal strains was carried out. The molecular identification by internal transcribed spacer (ITS) sequencing identified the strains as Aspergillus terreus, Aspergillus oryzae, Penicillium sp., Penicillium sp., Penicillium daleae and Penicillium sp. with GenBank accession numbers KP309809, KP309810, KP309811, KP309812, KP309813 and KP309814, respectively. The ability of the isolated fungal strains to tolerate and solubilize Mn was investigated by subculturing them on Mn-supplemented plates with concentration ranging from 0.1 to 0.5% (w/v). Mn solubilizing ability of the fungal isolates is possibly due to the mycelia production of biogenerated organic acids such as oxalic acid, citric acid, maleic acid and gluconic acid as revealed by ion chromatography. Our investigation signifies the role of fungi in biotransformation of insoluble Mn oxide.     

The role of 5-arylalkylamino- and 5-piperazino- moieties on the 7-aminopyrazolo[4,3-d]pyrimidine core in affecting adenosine A1 and A2A receptor affinity and selectivity profiles

New 7-amino-2-phenylpyrazolo[4,3-d]pyrimidine derivatives, substituted at the 5-position with aryl(alkyl)amino- and 4-substituted-piperazin-1-yl- moieties, were synthesized with the aim of targeting human (h) adenosine A₁ and/or A_2A receptor subtypes. On the whole, the novel derivatives 1–24 shared scarce or no affinities for the off-target hA_2B and hA₃ ARs. The 5-(4-hydroxyphenethylamino)- derivative 12 showed both good affinity (K_i =?150?nM) and the best selectivity for the hA_2A AR while the 5-benzylamino-substituted 5 displayed the best combined hA_2A (K_i =?123?nM) and A₁ AR affinity (K_i =?25?nM). The 5-phenethylamino moiety (compound 6) achieved nanomolar affinity (K_i =?11?nM) and good selectivity for the hA₁ AR. The 5-(N⁴-substituted-piperazin-1-yl) derivatives 15–24 bind the hA₁ AR subtype with affinities falling in the high nanomolar range. A structure-based molecular modeling study was conducted to rationalize the experimental binding data from a molecular point of view using both molecular docking studies and Interaction Energy Fingerprints (IEFs) analysis.     

Rhamnolipid production by a novel thermophilic hydrocarbon-degrading Pseudomonas aeruginosa AP02-1

Thermophilic bacterial cultures were isolated from a hot spring environment on hydrocarbon containing mineral salts media. One strain identified as Pseudomonas aeruginosa AP02-1 was tested for the ability to utilize a range of hydrocarbons both n-alkanes and polycyclic aromatic hydrocarbons as sole carbon source. Strain AP02-1 had an optimum growth temperature of 45°C and degraded 99% of crude oil 1% (v/v) and diesel oil 2% (v/v) when added to a basal mineral medium within 7 days of incubation. Surface activity measurements indicated that biosurfactants, mainly glycolipid in nature, were produced during the microbial growth on hydrocarbons as well as on both water-soluble and insoluble substrates. Mass spectrometry analysis showed different types of rhamnolipid production depending on the carbon substrate and culture conditions. Grown on glycerol, P. aeruginosa AP02-1 produced a mixture of ten rhamnolipid homologues, of which Rha-Rha-C₁₀-C₁₀ and Rha-C₁₀-C₁₀ were predominant. Rhamnolipid-containing culture broths reduced the surface tension to ≈28 mN and gave stable emulsions with a number of hydrocarbons and remained effective after sterilization. Microscopic observations of the emulsions suggested that hydrophobic cells acted as emulsion-stabilizing agents.     

Ganglioside alterations in YAC-1 cells cultivated in serum-supplemented and serum-free growth medium

Gangliosides of the G_M1b-pathway (G_M1b and GalNAc-G_M1b) have been found to be highly expressed by the mouse T lymphoma YAC-1 grown in serum-supplemented medium, whereas G_M2 and G_M1 (G_M1a-pathway) occurred only in low amounts [Müthing, J., Peter-Katalini, J., Hanisch, F.-G., Neumann, U. (1991)Glycoconjugate J 8:414–23]. Considerable differences in the ganglioside composition of YAC-1 cells grown in serum-supplemented and in well defined serum-free medium were observed. After transfer of the cells from serum-supplemented medium (RPMI 1640 with 10% fetal calf serum) to serum-free medium (RPMI 1640 with well defined supplements), G_M1b and GalNAc-G_M1b decreased and only low amounts of these gangliosides could be detected in serum-free growing cells. The expression of G_M1a was also diminished but not as strongly as that of G_M1b and GalNAc-G_M1b. These growth medium mediated ganglioside alterations were reversible, and the original ganglioside expression was achieved by readaptation of serum-free growing cells to the initial serum-supplemented medium. On the other hand, a new ganglioside, supposed to represent GalNAc-G_D1a and not expressed by serum-supplemented growing cells, was induced during serum-free cultivation, and increased strongly after readaptation. These observations reveal that the ganglioside composition ofin vitro cultivated cells can be modified by the extracellular environment due to different supplementation of the basal growth medium. Abbreviations: BSA, bovine serum albumin GSL(s), glycosphingolipid(s); HPTLC, high-performance thin-layer chromatography; LDL, low density lipoprotein; NeuAc,N-acetylneuraminic acid; NeuGc,N-glycoloylneuraminic acid. The designation of the following glycosphingolipids follows IUPAC-IUB recommendations. GgOse₃Cer or gangliotriaosylceramide, GalNAc1-4Gal1-4GlcCer; GgOse₄Cer or gangliotetraosylceramide, Gal1-3GalNAc1-4Gla1-4GlcCer; GgOse₅Cer or gangliopentaosylceramide, GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer; GgOse₆Cer or gangliohexaosylceramide, Gal1-3GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer or GgOse₆Cer; II³NeuAc-GgOse₃Cer or G_M2; II³NeuAc-GgOse₄Cer or G_M1 or G_M1a; IV³NeuAc-GgOse₄Cer or G_M1b; IV³NeuAc-GgOse₅Cer or GalNAc-G_M1b; IV³NeuAc-GgOse₆Cer or Gal-GalNAc-G_M1b; IV³NeuAc, II³NeuAc-GgOse₄Cer or G_D1a; II³(NeuAc)₂-GgOse₄Cer or G_D1b; IV³NeuAc, III⁶NeuAc-GgOse₄Cer or G_D1a; IV³NeuAc, II³NeuAc-GgOse₅Cer or GalNAc-G_D1a. Enzymes: Vibrio cholerae andArthrobacter ureafaciens neuraminidase (EC 3.2.1.18).     

Identification of suitable ionic liquids for application in the enzymatic hydrolysis of rutin by an automated screening

An automated method in milliliter scale was developed for the screening of process parameters concerning the hydrolysis of the flavonoid rutin catalyzed by the rhamnosidase activity of naringinase from Penicillium decumbens. Besides the effect of additives such as ionic liquids and low molecular salts, the productivity in a multiple phase system as well as the recyclability of the enzyme in repetitive batches were studied. The hydrophobic ionic liquid (IL) trihexyl(tetradecyl)phosphonium bis(trifluormethylsulfonyl)imide [P(h₃)t][Tf₂N] was identified to combine the most favorable characteristics out of 23 investigated ILs with regard to enzyme compatibility, substrate solubility and enzyme partition coefficient. Also, for the corresponding cations 1-ethyl-3-methylimidazolium [EMIM], 1-butyl-3-methylimidazolium [BMIM], 1-butyl-1-methylpyrrolidinium [BMPL] and 1-octyl-3-methylimidazolium [OMIM], the entity with the [Tf₂N] anion was best tolerated by the naringinase. With increasing IL content, higher space time yields with up to 1.5 g/(L h) for 80% (v/v) [P(h₃)t][Tf₂N] were achieved. Enhanced specific enzyme activity was observed in the presence of Ca²⁺ ions. By addition of [P(h₃)t][Tf₂N] and calcium chloride, the reactive aqueous phase was successfully used in three repetitive batches with full conversion.     

Synthesis of kyotorphin precursor from eutectic mixtures catalysed by α-chymotrypsin

The kyotorphin precursor, N-carbobenzoxyl-l-tyrosine-l-arginine amide (N-CBZ-l-Tyr-l-ArgNH₂), was synthesized from N-CBZ-l-tyrosine ethyl ester (N-CBZ-l-TyrOEt) and l-arginine amide (l-ArgNH₂) by using -chymotrypsin. Eutectic mixtures were formed by mixing the substrates in the presence of water and/or organic solvents as adjuvants. The eutectic temperature was obtained with a 0.45 l-ArgNH₂ mole fraction. It was lowered to 23 °C by addition of 10% (v/w) water and 5% (v/w) dimethyl formamide, thus maintaining homogeneous liquid states at the reaction temperature of 30 °C. After 9 h the solutions became solidified and no further reaction took place. Approximately 90% (mol/mol) conversion was achieved from the substrate mixtures with substrate contents responsible for more than 80% (w/w) of the total mixture.     

Cellular origin and distribution of transforming growth factor-β1 in the normal rat myocardium

Summary Transforming growth factor- (TGF-) is a biologically active polypeptide present in normal tissues as well as transformed cells. Two structurally related forms of this peptide are TGF- ₁ and TGF- ₂. Using freshly isolated cardiomyocytes and non-myocyte heart cells, and a [³²P]-labelled cDNA probe to human TGF- ₁, we demonstrated that mRNA for TGF- ₁ could be detected only in the nonmyocyte fraction of heart cells. In the present study, the distribution of TGF- ₁ in the heart was determined by immunofluorescence staining by use of a polyclonal antibody to porcine TGF- ₁ in cryostat sections of rat heart. Immunofluorescence staining was intense around the blood vessels and radially diffuse in the surrounding myocardium.     

Recovery of Cytokinins from Cation Exchange Resins

Cytokinins exhibit great affinity for the polystyrene matrix of Dowex-50 cation exchange resins. Model experiments showed that only c. 20% of the applied 2-methylthio-trans-zeatin and c. 70% of the trans-zeatin are eluted from AG 50W columns with six bed volumes of 1 N NH₄ OH. Inclusion of 70% (v/v) ethanol in the eluant increases the recovery to c. 55 and 85% respectively. No additional recovery was obtained upon continued elution to 14 bed volumes. Similar experiments with Duolite CS-101, a weak acid cation exchange resin with acrylic matrix, showed that c. 95% of the applied 2-methylthio derivatives of isopentenyladenine and zeatin are recovered from the resin with six bed volumes of 70% (v/v) ethanol (pH 10) or 1 N NH₄OH in 70% (v/v) ethanol. The less modified cytokinins, isopentenyladenosine and ribosylzeatin, are recovered completely under these conditions. Recovery of cytokinins was determined by means of high-pressure liquid chromatography of the column eluates.     

Adventitious root formation in Arabidopsis thaliana thin cell layers

This paper describes, for the first time, de novo adventitious root formation from thin cell layers (TCLs) of Arabidopsis thaliana. The objective of the study was to determine the optimal hormonal and light conditions and the optimal exogenous Ca²⁺ concentration for obtaining adventitious rooting (AR) from A. thaliana TCLs and to identify the tissue(s) involved in the process. The results show that maximum AR was obtained with a single-phase method in the presence of 10 M indole-3-butyric acid and 0.1 M kinetin under continuous darkness for 30 days and with 0.6 mM exogenous CaCl₂. The endodermis was the only tissue involved in root meristemoid formation. The role of Ca²⁺ in AR and the importance of using Arabidopsis TCLs in studies on the genetic/biochemical control of AR are discussed.Abbreviations AR Adventitious rooting - CIM Callus-inducing medium - Col-0 Columbia ecotype - 2,4-D 2,4-Dichlorophenoxyacetic acid - HFM Hormone-free medium - HM Medium with 10 M IBA and 0.1 M Kin - IBA Indole-3-butyric acid - Kin Kinetin - LS Longitudinal section - NAA -Naphthaleneacetic acid - RIM Root-inducing medium - TCL Thin cell layer - WS Wassilewskija ecotype     

Age-related increase of β1-adrenergic receptor gene expression in rat liver: a potential mechanism contributing to increased β-adrenergic receptor density and responsiveness during aging

In this study we examined whether the levels of gene expressions of the three β- adrenergic receptor (βAR) subtypes, β₁, β₂, and β₃, contribute to age-related increase in βAR density. Liver membranes and total RNA were prepared from young (4- to 6-month-old) and old (24-month-old) male Fischer 344 rats. βAR density (B_max) in liver membranes was measured by a radioligand receptor binding assay using the receptor subtype nonselective βAR antagonist ¹²⁵I-pindolol as the radioligand. Steady-state levels of β₂AR mRNA in rat liver were measured by Northern blot analysis; because of the low abundance of β₁AR and β₃AR mRNA in rat liver, the expressions of these genes were measured by a semiquantitative RT-PCR or an RT-PCR. Scatchard analysis of saturation binding curves of the binding assay confirmed an age-related increase in B_max (young: 7.1?±?0.8?fmol/mg protein vs. old: 18.1?±?4.3?fmol/mg protein). No age-related differences were found in the levels of β₂AR mRNA. However, semiquantitative RT-PCR revealed an approximately twofold increase in β₁AR mRNA level between young and old rats (P?<?0.05). β₁AR mRNA levels were also correlated with B_max values for ¹²⁵I-pindolol binding sites in individual rats (r = 0.67; P?=?0.012). β₃AR mRNA, which was demonstrable in rat white adipose tissue by RT-PCR, was generally not detected in livers from young or old rats, with the exception of two old rats with the highest B_max. These results suggest that an age-related increase of β₁AR gene expression contributes to increased βAR density and β adrenergic responsiveness in rat liver during aging.     

Low night temperature effect on photosynthate translocation of two C4 grasses

Summary Translocation of assimilates in plants of Echinochloa crus-galli, from Quebec and Mississippi, and of Eleusine indica from Mississippi was monitored, before and after night chilling, using radioactive tracing with the short-life isotope ¹¹C. Plants were grown at 28°/22°C (day/night temperatures) under either 350 or 675 l·l^-1 CO₂. Low night temperature reduced translocation mainly by increasing the turn-over times of the export pool. E. crus-galli plants from Mississippi were the most susceptible to chilling; translocation being completely inhibited by exposure for one night to 7°C at 350 l·l^-1 CO₂. Overall, plants from Quebec were the most tolerant to chilling-stress. For plants of all three populations, growth under CO₂ enrichment resulted in higher ¹¹C activity in the leaf phloem. High CO₂ concentrations also seemed to buffer the transport system against chilling injuries.     

Growth of food-borne pathogenic bacteria in oil-in-water emulsions: II—Effect of emulsion structure on growth parameters and form of growth

The growth rates and yields of Listeria monocytogenes and Yersinia enterocolitica were determined in liquid culture media, and in model oil-in-water emulsions that contained 30, 70 or 83% (v/v) hexadecane. In emulsions with a mean droplet size of 2 μm containing 83% (v/v) hexadecane, the growth of both organisms resulted in decreased yields. Additionally, in these emulsions adjusted to pH 5·0 or 4·4 the growth rate of L. monocytogenes was significantly less than in other model systems which had an aqueous phase of equivalent chemical composition. Microscopic examination of the 83% (v/v) emulsion showed that its microstructure immobilized the bacteria, which were constrained to grow as colonies. Bacteria behaved similarly in model emulsions of either hexadecane or sunflower oil. Manipulation of the droplet size distribution of the emulsions changed the form and rate of growth of bacteria within them.     

INVESTIGATION OF THE GIBBERELLIC ACID OPTIMIZATION WITH A STATISTICAL TOOL FROM Penicillium variable IN BATCH REACTOR

The culture conditions for gibberellic acid (GA₃) production by the fungus Penicillium variable (P. variable) was optimized using a statistical tool, response surface methodology (RSM). Interactions of culture conditions and optimization of the system were studied using Box–Behnken design (BBD) with three levels of three variables in a batch flask reactor. Experimentation showed that the model developed based on RSM and BBD had predicted GA₃ production with R ²  = 0.987. The predicted GA₃ production was optimum (31.57 mg GA₃/kg substrate) when the culture conditions were at 7 days of incubation period, 21% v/w of inoculum size, and 2% v/w of olive oil concentration as a natural precursor. The results indicated that RSM and BBD methods were effective for optimizing the culture conditions of GA₃ production by P. variable mycelia.     

Biohydrogen production from wheat straw hydrolysate by dark fermentation using extreme thermophilic mixed culture

Hydrolysate was tested as substrate for hydrogen production by extreme thermophilic mixed culture (70°C) in both batch and continuously fed reactors. Hydrogen was produced at hydrolysate concentrations up to 25% (v/v), while no hydrogen was produced at hydrolysate concentration of 30% (v/v), indicating that hydrolysate at high concentrations was inhibiting the hydrogen fermentation process. In addition, the lag phase for hydrogen production was strongly influenced by the hydrolysate concentration, and was prolonged from approximately 11 h at the hydrolysate concentrations below 20% (v/v) to 38 h at the hydrolysate concentration of 25% (v/v). The maximum hydrogen yield as determined in batch assays was 318.4 ± 5.2 mL‐H₂/g‐sugars (14.2 ± 0.2 mmol‐H₂/g‐sugars) at the hydrolysate concentration of 5% (v/v). Continuously fed, and the continuously stirred tank reactor (CSTR), operating at 3 day hydraulic retention time (HRT) and fed with 20% (v/v) hydrolysate could successfully produce hydrogen. The hydrogen yield and production rate were 178.0 ± 10.1 mL‐H₂/g‐sugars (7.9 ± 0.4 mmol H₂/g‐sugars) and 184.0 ± 10.7 mL‐H₂/day L_reactor (8.2 ± 0.5 mmol‐H₂/day L_reactor), respectively, corresponding to 12% of the chemical oxygen demand (COD) from sugars. Additionally, it was found that toxic compounds, furfural and hydroxymethylfurfural (HMF), contained in the hydrolysate were effectively degraded in the CSTR, and their concentrations were reduced from 50 and 28 mg/L, respectively, to undetectable concentrations in the effluent. Phylogenetic analysis of the mixed culture revealed that members involved hydrogen producers in both batch and CSTR reactors were phylogenetically related to the Caldanaerobacter subteraneus, Thermoanaerobacter subteraneus, and Thermoanaerobacterium thermosaccharolyticum. Biotechnol. Bioeng. 2010;105: 899–908. © 2009 Wiley Periodicals, Inc.     

Glutamate-immunoreactivity in the trigeminal and dorsal root ganglia, and intraspinal neurons and fibres in the dorsal horn of the rat

Summary Putative aspartergic and glutamatergic sensory neurons in the rat were identified by autoradiography and immunocytochemistry respectively. Approximately 3% of large L₄ dorsal root ganglion neurons (diameter 18–52 m) accumulated radiolabelled aspartate, whereas all satellite glia had high affinity for the amino acid. Glutamate-immunofluorescent (Glu-FITC) dorsal root ganglia neurons comprised 38.3% at S₁, 35.6% at L₂ 33.9% at C₅ and 28.8% at T₆. Numbers of immunoreactive neurons were higher with the more sensitive peroxidase-anti-peroxidase (Glu-PAP) method; and the cell counts totalled 42% (S₁), 41.2% (L₄), 35% (C₅) and 34.6% (T₆). The trigeminal ganglion (TG) contained 24% Glu-FITC and 32.3% Glu-PAP positive cells. The majority of glutamate-immunoreactive sensory neurons were small, ranging from 10–35 m with median diameters of 17.5m (C₅), 21m (S₁), 24.2m (TG) and 28.5 m (L₂). It is evident therefore, that a subgroup of class B cells are glutamatergic. Glutamate immunoreactivity in the spinal cord was similar in all segments and was localized in the superficial lamina and substantia gelatinosa of the dorsal horn. Stained interneurons were located among the immunoreactive fibres. The dorsolateral funiculus contained dense plexus of immunoreactive fibres which increased in prominence after intraperitoneal injection of L-glutamate, but penetration of exogenous glutamate into the grey matter was limited. Instead, the meninges and basal layers of the spinal blood vessels were intensely immunoreactive. The studies describe the subtypes of acidic amino acidergic neurons and relates the immunohistochemistry to a functional subclass.     

Preparation and some properties of immobilized Penicillium funiculosum 258 dextranase

The production of dextranase was investigated in static cultures of Penicillium funiculosum 258. Maximal enzyme productivity was attained at pH 8.0, with 3.5% (w/v) dextran (MW, 260 000) as carbon source, NaNO₃ (1%, w/v) and yeast extract (0.2%, w/v) as nitrogen source, 0.4% (w/v) K₂HPO₄ and 0.06% (w/v) MgSO₄. It was possible to increase the productivity of dextranase to 41.8 units ml⁻¹ in the modified medium. The enzyme was immobilized on different carriers by different techniques of immobilization. The enzyme prepared by covalent binding on chitosan using glutaraldehyde had the highest activity, the immobilized enzyme retaining 63% of its original specific activity. Compared with the free dextranase, the immobilized enzyme exhibited: a higher pH optimum, a higher optimal reaction temperature and energy of activation, a higher Michaelis constant, improved thermal stability and higher values of deactivation rate constant. The immobilized enzyme retained about 80% of the initial catalytic activity even after being used for 12 cycles.