Immobilization of Lipase from Mucor Miehei Onto Poly (Ethylene) Based Graft Copolymers

Lipase from Mucor miehei was immobilized covalently onto hydrolyzed poly(ethylene)-g.co-hydroxyethyl methacrylate (PE-HEMA). This hydrolysis of the copolymer was achieved using 0.1 M NaOH over different periods of time, under controlled conditions. The graft copolymers and their hydrolyzed equivalents were characterized by scanning electron microscopy (SEM) and by differential scanning calorimetry analysis (DSC). Water sorption studies were undertaken to provide a measure of relative hydrophobicity of the samples.

The lipase immobilization reaction was studied in order to assess the effects of controlling various important parameters. These include the nature of the buffering medium, the time over which the immobilization was allowed to occur, the concentration of the activating and coupling agent used (CMC) and the concentration of enzyme employed during attempts at effective immobilization. The immobilized lipase was used in the hydrolysis of triolein (glycerol trioleate). From this study, the apparent K_M, the optimum pH for hydrolysis and the optimum temperature for hydrolysis were revealed.

The suitability of hydrolyzed poly(ethylene)-g.co-HEMA as a support in the immobilization of lipase was assessed by determination of the amount of lipase coupled to the support and by assessment of the retention of activity of the immobilized lipase after its exposure to the immobilization reagents, procedure and conditions.     

Burrowing beetles of the genus Bledius (Staphylinidae) as agents of bioturbation in the emergent areas and shores of an athalassic inland lake (Fuente de Piedra,southern of Spain)

Bledius (Elbidus) bicornis (Germ.) and B. (Eucerotobledius) furcatus (Oliv.) (Coleoptera: Staphylinidae) are the most important burrowing species in the emergent areas and shores in the athalassic lake of Fuente de Piedra (Málaga, S. of Spain). A first estimate of the importance of these organisms in this system is presented. These insects kick out sediment during their burrowing activity, which accumulates on the surface near the burrows as tumuli which can be easily eroded. The lake perimeter (17 km) is densely colonized (usual densities from 1700 to 2500 ind m⁻²). The amount of granulated material that can be potentially kicked out was 46.22 g dry wt m⁻² day⁻¹. At the same time, the material that constitutes the tumuli shows different characteristics from the compact ground below the surface. Thus, it is relatively enriched with organic matter (6.15 g per square meter), soluble phosphate (406.5 μg m⁻²) and ammonium (4856 μg m⁻²), whereas it lacks nitrate. Results of a transect from uninhabited areas to zones of maximum population density also show a similarity between the higher ground level of ammonium and phosphate concentrations and population density.     

The growth and degeneration of the hydroid Sertularia perpusilla,an obligate epiphyte of leaves of the seagrass Posidonia oceanica

The hydrorhizae of Sertularia perpusilla colonies that originate from stolon attachments initially grow up and down Posidonia leaves. After a short time the distal hydroid tissue degenerates concurrently with the downward growth of the proximal hydrorhiza onto younger sections of the leaves. Consequently the hydroids move down the leaves which grow upward from a basal meristem. In situ inversion of leaves had no effect on either the orientation or the rates of growth and degeneration. This indicates that orientation may be in response to age related features of leaf tissue rather than to light, gravity or water movement, the directions of which were reversed by inverting the leaves. However, orientation of growth of new hydroids from attached stolons is immediate, probably before the hydroid colony is long enough to be able to detect an age gradient. This paradox could be explained if initial orientation were based on the direction of gravity or light but subsequent growth were directed along the age gradient. For hydroids in the size range studied there was a relation between growth rate and number of hydranths. Thus growth rate may be limited by food rather than by there being only one growing tip of the hydrorhizae.     

The product of the nitrogen fixation regulatory gene nfrX of Azotobacter vinelandii is functionally and structurally homologous to the uridylyltransferase encoded by glnD in enteric bacteria.

We sequenced the nitrogen fixation regulatory gene nfrX from Azotobacter vinelandii, mutations in which cause a Nif- phenotype, and found that it encodes a 105-kDa protein (NfrX), the N terminus of which is highly homologous to that of the uridylyltransferase-uridylyl-removing enzyme encoded by glnD in Escherichia coli. In vivo complementation experiments demonstrate that the glnD and nfrX products are functionally interchangeable. A vinelandii nfrX thus appears to encode a uridylyltransferase-uridylyl-removing enzyme, and in this paper we report the first sequence of such a protein. The Nif- phenotype of nfrX mutants can be suppressed by a second mutation in a recently identified nifL-like gene immediately upstream of nifA in A. vinelandii. NifL mediates nif regulation in response to the N status in A. vinelandii, presumably by inhibiting NifA activator function as occurs in Klebsiella pneumoniae; thus, one role of NfrX is to modify, either directly or indirectly, the activity of the nifL product.     

Seasonal fluctuations of zooplankton biomass in Lake Xolotlán (Managua)

Seasonal fluctuations of zooplankton biomass (dry weight) were determined during a year in two localities of Lake Xolotlán (Managua). Biomass estimations of the most common species of rotifers, cladocerans and copepods were made. The maximal zooplankton biomass was observed in February–April (dry season) in coincidence with the period of highest phytoplankton abundance. Copepods contributed with 78% and 84% to the mean zooplankton biomass at points 1 and 7, respectively. Cladocera biomass was lowest during most of the year, and it was probably controlled by fish predation. Development of rotifer biomass was more intense during the rainy season, when detritus particles were more abundant. Daily fluctuations of zooplankton biomass were not pronounced.     

Clostridium sticklandii glycine reductase selenoprotein A gene: cloning, sequencing, and expression in Escherichia coli.

Gene grdA, which encodes selenoprotein A of the glycine reductase complex from Clostridium sticklandii, was identified and characterized. This gene encodes a protein of 158 amino acids with a calculated M(r) of 17,142. The known sequence of 15 amino acids around the selenocysteine residue and the known carboxy terminus of the protein are correctly predicted by the nucleotide sequence. An opal termination codon (TGA) corresponding to the location of the single selenocysteine residue in the polypeptide was found in frame at position 130. The C. sticklandii grdA gene was inserted behind the tac promotor of an Escherichia coli expression vector. An E. coli strain transformed with this vector produced an 18-kDa polypeptide that was not detected in extracts of nontransformed cells. Affinity-purified anti-C. sticklandii selenoprotein A immunoglobulin G reacted specifically with this polypeptide, which was indistinguishable from authentic C. sticklandii selenoprotein A by immunological analysis. Addition of the purified expressed protein to glycine reductase protein components B and C reconstituted the active glycine reductase complex. Although synthesis of enzymically active protein A depended on the presence of selenium in the growth medium, formation of immunologically reactive protein did not. Moreover, synthesis of enzymically active protein in a transformed E. coli selD mutant strain indicated that there is a nonspecific mechanism of selenocysteine incorporation. These findings imply that mRNA secondary structures of C. sticklandii grdA are not functional for UGA-directed selenocysteine insertion in the E. coli expression system.     

Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor: part I. lipase adsorption studies

Adsorption of proteins from a crude preparation containing a lipase from Aspergillus niger on microporous polypropylene hollow fibers was studied at six different temperatures. Langmuir isotherms accurately describe the overall adsorption equilibria. Lipase is selectively adsorbed relative to the other proteins in the crude preparation. Hence, immobilization also provides further purification of the lipase. The predictions of the Langmuir model for the change in the specific activity of lipase upon adsorption are consistent with experimental results. The loading capacity of the hollow fibers decreases and the adsorption constant increases as temperature is increased. This effect is more significant in the case of lipolytic activity than it is for the total amount of adsorbed protein. Small, positive enthalpy changes are associated with the adsorption of lipase on these hydrophobic membranes.     

Amino acid substitutions at tryptophan 388 and tryptophan 412 of the HepG2 (Glut1) glucose transporter inhibit transport activity and targeting to the plasma membrane in Xenopus oocytes.

All 6 tryptophan residues in the human HepG2-type glucose transporter (Glut1) were individually altered by site-directed mutagenesis to investigate the role of these residues in transport function. Tryptophan residues in positions 48, 65, 186, 363, 388, and 412 of Glut1 were changed to either a glycine or leucine residue. Mutant mRNAs were synthesized and injected into Xenopus laevis oocytes. Transporter function as assessed by uptake of 2-deoxy-D-[3H]glucose or transport of 3-O-[3H]methylglucose was decreased in the 388 and 412 mutants but was unaltered in all other mutants. The amount of the mutant transporters expressed in total membrane and plasma membrane fractions was measured using Glut1-specific antibodies. Calculation of the intrinsic transport activity of each of the mutants using these data demonstrated that the reduced transport activity of the 412 mutants was caused entirely by a dramatic decrease in the intrinsic activity of the mutant proteins whereas the reduced activity of the 388 mutants was a result of a decreased level of the protein in oocytes, decreased targeting to the plasma membrane, and a modest decrease in the intrinsic activity. Protease/glycosidase mapping of in vitro translation products indicated that the effects of the 388 and 412 point mutations could not be attributed to a disruption in the ability of the mutant proteins to insert properly into the membrane. The ID50 for cytochalasin B inhibition of 2-deoxyglucose uptake was increased from 5 x 10(-7) M for the wild-type Glut1 to 4 x 10(-6) M in the 388 mutants but was unaltered in the 412 mutants. These observations suggest that 1) Trp-412 may comprise part of a hexose binding site or is involved in maintaining a local tertiary structure critical for transport function; 2) Trp-388 is involved in stabilizing the equilibrium binding of cytochalasin B to the transporter. Trp-388 may therefore lie near a substrate binding site and also appears to participate in stabilization of local tertiary structure important for full catalytic activity and efficient targeting to the Xenopus plasma membrane.     

The influence of irradiance on growth, photosynthesis and respiration of Gyrodinium cf. aureolum

The bloom-forming marine dinoflagellate Gyrodinium cf. aureolumwas grown in batch cultures over a range of irradiances (35–380µmolm^–2 s^–1 and growth, photosynthesis and respirationrates determined. Saturation of growth occurred at irradiancesof 100µmol m^–2 s^–1 Below this light level,decreases in growth rates and cell size, and a relative increasein carbon specific respiration rates, were observed. On theother hand, photosynthesis-irradiance relationships determinedfrom dissolved oxygen incubations showed that on a cellularand carbon basis, cultures grown at low irradiances had higherrates of light-limited and light-saturated photosynthesis, mainlyas a result of large increases in cell chlorophyll content.This adaptation strategy enables low-light-grown organisms toexploit available high irradiance through a relatively highphotosynthetic capacity. In cells grown at higher light levels(>100µmol m^–2 s^–1), excess photosynthatemay be diverted to storage rather than used for growth.     

Ada software for cytometry.

Ada is a new general-purpose language that embodies the concepts of software engineering. Although it was initially developed for military purposes, it is suitable for developing software for cytometry and other health-related applications. A pilot study has demonstrated the feasibility of employing Ada for cytometry applications. Three packages were created. The first subtracts a control three-dimensional population from multiple individual experimental populations and presents the results in spread sheet form. A second package has the capability of finding aggregates of cells. The results of this package are visualized employing a commercially available program for three-dimensional presentation of the data that permits rotation in real time. A third package consists primarily of interface drivers for two commercially available personal computer boards, an ADC and a stepper motor controller. The major problems with the coding were due to incomplete implementation of the language. This pilot study, together with others, indicates that it would be both cost effective and beneficial to implement cytometry and other medical devices in Ada.