Monoclonal antibody purification by affinity chromatography with ligands derived from the screening of peptide combinatory libraries

The peptide, Ala-Pro-Ala-Arg (APAR), was selected from the screening of a tetrapeptide combinatorial synthetic library as the ligand for affinity purification of an anti-Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) monoclonal antibody (Mab) developed in mouse ascitis. The affinity chromatographic matrix obtained by attachment of APAR to agarose, having a peptide density of 0.5 mol ml^–1, showed a maximum capacity of 9.1 mg Mab ml^–1 and a dynamic capacity of 3.9 mg Mab ml^–1. A 95% yield of electrophoretically pure anti-GM-CSF was obtained in a single step.     

Examining the relationship between bacteria and heterotrophic nanoflagellates in Funka Bay (Japan) using the size-fractionation method

The chemical and biological conditions, and the bacteria-heterotrophic nanoflagellate (HNF) relationship were investigated in the vicinity of Funka Bay, southwest of Hokkaido, Japan during early spring 1999. At the time of sampling, chlorophyll a concentration, bacteria, phycoerythrin rich-cyanobacteria, and HNF abundance were in the following ranges: 0.3–3.6 g l^–1, 2.5–5.6 × 10⁵ cells ml^–1, 0.6–1.2 × 10³ cells ml^–1, and 2.2–4.2 × 10³ cells ml^–1, respectively. Dissolved inorganic nitrogen, phosphate and silicate concentrations were in the ranges: 8.7–12.2 M, 0.9–2.0 M, and 21.6–25.5 M, respectively. Primary production ranged from 6.4 to 76.3 mg C m^–3 d^–1. Using water samples from regions of different productivity levels (in and outside bay), the bacteria - HNF relationship was uncoupled experimentally by the size-fractionation technique. Higher primary production (19.9 mg C m^–3 d^–1) in the bay supported higher bacterial growth rate (0.029 h^–1). However, outside the bay both primary production (6.4 mg C m^–3 d^–1) and bacterial growth rate (0.007 h^–1) were lower. The HNF growth rates and grazing rates were similar for both but by comparing both HNF grazing capacity and bacterial production, there was net decrease in bacterial abundance outside the bay and net increase inside the bay. The microbial parameters (rates and abundance) and the amount of carbon flow estimated through the phytoplankton – dissolved organic matter (DOM) – bacteria loop were different between the coastal station and the open ocean station. However HNF grazing and growth rates was similar for both stations.     

Optical and electrical studies on dansyllysine-valinomycin in thin lipid membranes

Summary Dansyllysine-valinomycin, a fluorescent analogue of the ionophore valinomycin was synthesized and incorporated into black lipid membranes. Its concentration inside the membrane was measured fluorometrically and was also determined from electrical relaxation experiments, which were analyzed on the basis of a previously proposed carrier model. The results of both methods agreed within less than one order of magnitude. This appears satisfactory in view of the sources of error inherent in both procedures.A conductance increment per carrier molecule of about 3 · 10^{–17 –1} was obtained for dansyllysine-valinomycin in diphytanoyllecithin membranes at 25 C and 1M RbCl in the aqueous phases. This is about 400 times smaller compared to unmodified valinomycin in monoolein membranes. The difference is mainly caused by the change in the membrane properties and to a smaller extent by the structural modification of the carrier.     

Thermostable cellulases from <Emphasis Type="Italic">Streptomyces</Emphasis>sp.: scale-up production in a 50-l fermenter

Thermostable cellulase was produced by Streptomyces sp. T3-1 grown in a 50-l fermenter. Maximum cellulase activity was attained on the fourth day when agitation speeds and aeration rates were controlled at 300 rpm and 0.75 vvm, respectively. Maximum enzyme activities were: 148 IU CMCase ml^–1, 45 IU Avicelase ml^–1, and 137 IU -glucosidase ml^–1 with productivity of 326 IU l^–1 h^–1, which were 10--32% higher than the values obtained in shake-flask culturesRevisions requested 12 October 2004/1 November 2004; Received received 1 November 2004/14 December 2004     

Enhancement of production of cloned α-amylase by lactic acid feeding from recombinant Saccharomyces cerevisiae using a SUC2 promoter

-Amylase production was higher (13 units ml^–1) when a recombinant Saccharomyces cerevisiae containing a SUC2 promoter was grown with 10 g lactic acid l^–1 than without addition (8 units ml^–1). With continuous lactic acid feeding in the inducing phase, -amylase increased to 79 units ml^–1 in a 1-l jar fermenter.     

Quantification of the contribution of surface outgrowth to biocatalysis in sol-gels: oxytetracycline production by <Emphasis Type="Italic">Streptomyces rimosus</Emphasis>

A technique was developed for differentiating the activity of microbes solely within sol gels by using the contribution of biomass outgrowth. Streptomyces rimosus was immobilised in colloidal silica gels and biomass growth, oxytetracycline synthesis, pH and carbohydrate consumption were compared for UV surface-sterilised gels, untreated gels, and liquid cultures. Absolute and biomass specific oxytetracycline yields were higher for non-sterile gels than for liquid culture. Biomass solely within colloidal silica gels (1.7 mg ml^–1), and gels obtained from colloidal silica modified by addition of larger silica particles (1.2 mg ml^–1) yielded 27 and 21 g ml^–1 oxytetracycline compared with 97 and 104 g ml^–1 for unsterilised gels (3.6 and 5.2 mg ml^–1 biomass) displaying outgrowth. It was therefore apparent that biomass and antibiotic production within the gels was limited and that optimisation requires gel modification.     

The effects of aflatoxin B1 on immature germinating maize (Zea mays) embryos

Immature maize (Zea mays L.) embryos were treated with aflatoxin B₁ concentrations, ranging from 0.1 g ml^–1 to 25 g ml^–1. Below 5 g ml^–1 aflatoxin B₁, root and shoot elongation was not significantly inhibited. Ultrastructurally, root tip cells showed little deterioration, except a possible diffused clearing in mitochondria and plastids. As the toxin concentration was increased above 5 gml^–1, shoot, and particularly root elongation, was progressively inhibited. Associated with this, there was an apparent decrease in the ribosome population. Furthermore, membranes, particularly the vacuolar membrane, became abnormal and vacuolar distension occurred. At 20 and 25 g ml^–1, these effects were exacerbated, and mitochondria and plastid structure was disrupted. At these concentrations, there was evidence of a disruption in lipid metabolism. The results are discussed in the context of known aflatoxin effects on cellular control mechanisms and ultrastructure in animal systems.     

Preparation of vinyl thymidine ester catalyzed by protease and its chemical polymerization

Transesterification reaction of 0.25 M thymidine with 1 M divinyladipate in dimethylformamide (DMF) was catalyzed by an alkaline protease (5 mg ml^–1) from Streptomyces sp. (20 units mg^–1 min) at 30 °C for 7 days to give 5-O-vinyladipoyl thymidine (yield 77%) without formation of any by-products. Poly(vinyl alcohol) containing thymidine branches could be obtained by its free-radical polymerization.     

The relative importance of different ciliate taxa in the pelagic food web of lake constance

Abundance, biovolume, and species composition of pelagic ciliates in Lake Constance were recorded over two annual cycles (1987/88). Production was estimated from mean annual biovolumes and size-specific growth rates obtained from the literature. Cell concentrations and biovolumes ranged from 0.1 to 120 cells ml^–1 and from 3 to 1,200 mm³ m^–3, respectively. Mean annual values were, respectively, 6.8 cells ml^–1 and 94 mm³ m^–3 in 1987, and 12.0 cells ml^–1 and 130 mm³ m^–3 in 1988. In both years, prostome nanociliates (<20m) dominated numerically, while strobiliids in the size range 20–35m contributed most significantly to ciliate production. Ciliate community production, according to a crude calculation, yielded approximately 10–15 g C m^–2 year^–1.     

Biosorption of uranium(VI) by Aspergillus fumigatus

Aspergillus fumigatus removed uranium(VI) very rapidly and reached equilibrium within 1 h of contact of biomass with the aqueous metal solution. Biosorption data fitted to Langmuir model of isotherm and a maximum loading capacity of 423 mg U g^–1 dry wt was obtained. Distribution coefficient as high as 10,000 (mg U g^–1)/(mg U ml^–1) at a residual metal ion concentration of 19 mg l^–1 indicates its usefulness in removal of uranium(VI) from dilute waste streams. Optimum biosorption was seen at pH 5.0 and was independent of temperature (5–50°C ). Initial metal ion concentration significantly influenced uptake capacity which brought down % (w/w) uranium(VI) removal from 90 at 200 mg U l^–1 to 35 at 1000 mg U l^–1. Presence of 0.84 mmol Fe²⁺, Fe³⁺, Ca²⁺ and Zn²⁺ had no effect on uranium(VI) biosorption unlike Al³⁺ (0.84 mM) which was inhibitory.     

Phenotypic expression of Vogesella indigofera upon exposure to hexavalent chromium,Cr6+

A eubacterium producing a blue pigment was isolated from a drinking water filter, and subsequently identified as Vogesella indigofera. This bacterium was further investigated for its morphological and biochemical characteristics after exposure to hexavalent chromium, Cr⁶⁺. The threshold Cr⁶⁺ concentration inhibiting the pigment production by V. indigofera was 200–300 g ml^–1 in liquid cultures of nutrient broth and 100–150 g ml^–1 on nutrient agar plates. The Cr⁶⁺ concentration preventing V. indigofera growth was 300–400 g ml^–1 in liquid cultures, but greater than 150 g ml^–1 on agar plates. Moreover, rugose colonies without the blue pigmentation were observed on agar plates amended with 150 (g Cr⁶⁺) ml^–1. The biochemical utilization profiles of the colonies without pigmentation did not differ from the original pigment-producing ones, indicating phenotypic plasticity of this bacterium. The difference of phenotypic expression of V. indigofera under various Cr⁶⁺ concentrations might have potential application as a pollution bioindicator for heavy metals.     

Expansion and neural differentiation of embryonic stem cells in adherent and suspension cultures

The embryonic stem cell line, S25, is a genetically modified line that allows lineage selection of neural cells (M. Li, L. Lovell-Badge, A. Smith (1998) Current Biology 8: 971–974). Here, the growth parameters of this cell line were analysed. Serial passaging in adherent conditions enabled these cells to grow rapidly (average specific growth rates of 0.035 h^–1) and generate high viable cell densities (above 90%). The aggregation of the S25 cells into embryoid bodies (EBs) was also studied, indicating limited cell growth (maximum cell densities of 2.7×10⁵ cells ml^–1) and a high variability of aggregate size (70–400 m after 8 d). Enzymatic dissociation of EBs with 1% (v/v) trypsin gave highest cell viability (91%) and density (1.4×10⁴ cells ml^–1) and the cells thus obtained are able to differentiate into neurons.     

Cadmium interferes with steroid biosynthesis in rat granulosa and luteal cellsin vitro

Recently, cadmium has been described to disturb ovarian function in rats. In this paper the direct influence of cadmium on steroid production of ovarian cellsin vitro has been studied. Granulosa and luteal cells were obtained from proestrous and pregnant rats, and incubated with 0, 5, 10, 20 or 40 g ml^–1 CdCl₂ in the presence or absence of 0.1–1000 ng ml^–1 follicle stimulating hormone (FSH) or luteinizing hormone (LH) for 24 or 48 h. Production of progesterone (P) and 17-estradiol (E₂) by granulosa and that of P by luteal cells were measured by radioimmunoassay. In FSH-stimulated granulosa cell cultures, 5 and 40 g ml^–1 CdCl₂ suppressed P accumulation to 65 and 10%, respectively; accumulation of E₂ (at 5 g ml^–1 CdCl₂) decreased to 44%. P production of LH-supported luteal cells dropped to 86 and 66%, respectively, when 5 and 40 g ml^–1 CdCl₂ was added to the medium. No alteration in basal P accumulation occurred in granulosa and luteal cell cultures following incubations with 20 and 40 g ml^–1 CdCl₂, whereas basal E₂ production of granulosa cells was markedly diminished. It is concluded that CdCl₂ suppressing steroid synthesisin vitro exerts a direct influence on granulosa and luteal cell function.     

Momordica charantia fruit juice stimulates glucose and amino acid uptakes in L6 myotubes

The fruit of Momordica charantia (family: Cucurbitacea) is used widely as a hypoglycaemic agent to treat diabetes mellitus (DM). The mechanism of the hypoglycaemic action of M. charantia in vitro is not fully understood. This study investigated the effect of M. charantia juice on either ³H-2-deoxyglucose or N-methyl-amino-a-isobutyric acid (¹⁴C-Me-AIB) uptake in L6 rat muscle cells cultured to the myotube stage. The fresh juice was centrifuged at 5000 rpm and the supernatant lyophilised. L6 myotubes were incubated with either insulin (100 nM), different concentrations (1–10 g ml^–1) of the juice or its chloroform extract or wortmannin (100 nM) over a period of 1–6 h. The results were expressed as pmol min^–1 (mg cell protein)^–1, n= 6–8 for each value. Basal ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes by L6 myotubes after 1 h of incubation were (means ± S.E.M.) 32.14 ± 1.34 and 13.48 ± 1.86 pmol min^–1 (mg cell protein)^–1, respectively. Incubation of L6 myotubes with 100 nM insulin for 1 h resulted in significant (ANOVA, p < 0.05) increases in ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes. Typically, ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes in the presence of insulin were 58.57 ± 4.49 and 29.52 ± 3.41 pmol min^–1 (mg cell protein^–1), respectively. Incubation of L6 myotubes with three different concentrations (1, 5 and 10 g ml^–1) of either the lyophilised juice or its chloroform extract resulted in time-dependent increases in ³H-deoxy-D-glucose and ¹⁴C-Me-AIB uptakes, with maximal uptakes occurring at a concentration of 5 g ml^–1. Incubation of either insulin or the juice in the presence of wortmannin (a phosphatidylinositol 3-kinase inhibitor) resulted in a marked inhibition of ³H-deoxyglucose by L6 myotubes compared to the uptake obtained with either insulin or the juice alone. The results indicate that M. charantia fruit juice acts like insulin to exert its hypoglycaemic effect and moreover, it can stimulate amino acid uptake into skeletal muscle cells just like insulin. (Mol Cell Biochem 261: 99–104, 2004)     

High density culture of the freshwater rotifer,Brachionus calyciflorus

The freshwater rotifer, Brachionus calyciflorus is one of the live food organisms used for the mass production of larval fish. In this study possibility of obtaining high density cultures of the freshwater rotifer B. calyciflorus were investigated. The two culture systems used differed in their air and dissolved oxygen supplies using three temperatures in each case: 24, 28 and 32 °C. Rotifers were batch-cultured using 5 l-vessels and fed with the freshwater Chlorella. The growth rate of rotifers significantly increased with an increase in temperature. The maximum density of the rotifers with air-supply at 24 °C, 6500 ind. ml^–1, was significantly lower than those cultured at 28 and 32 °C, i.e. 8600 and 8100 ind. ml^–1, respectively. Dissolved oxygen levels decreased with time and ranged from 0.8 to 1.4 mg l^–1 when the density of freshwater rotifer was the highest at each temperature. The highest density (19200 ind. ml^–1) of freshwater rotifer was obtained in cultures with a supply of oxygen at 28 °C. Densities of 13500 and 17200 ind. ml^–1 were found at 24 and 32 °C, respectively. Levels of NH₃-N increased with time and a dramatic increase of NH₃-N was observed at high temperatures. Levels of NH₃-N at 24, 28 and 32 °C were 13.2, 18.5 and 24.5 mg l^–1, respectively. These levels coincided with the highest rotifer density at each of the three temperatures. When rotifers were cultured with an oxygen-supply and pH was adjusted to 7, the maximum density of rotifer reached 33500 ind. ml^–1 at 32 °C . These results suggested that high density culture of freshwater rotifer, B. calyciflorus could be achieved under optimal conditions with DO value of exceeding 5 mg l^–1 and NH₃-N values of lower than 12.0 mg l^–1.     

Substitution of D-Trp32 in NPY Destabilizes the Binding Transition State to the Y1 Receptor Site in SK-N-MC Cell Membranes

The retention rate of the spin label 3-isothiocyanto methyl-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl spin label (proxyl) attached to the porcine N-acetyl-NPY peptide and the porcine N-acetyl-D-Trp³²-NPY peptide at Lys⁴ was investigated using SK-N-MC neuroblastoma cell membranes containing the Y1 receptor. The release rate of the spin labeled peptides was monitored by electron spin resonance and the K_D was determined by a direct radiolabeled NPY displacement binding assay. The analyses show that for the porcine [Ac-Tyr¹N⁴-proxyl]-NPY, the K_D was 8 × 10^–10 M and k_off was 2.7 × 10^–4 sec^–1 yielding a value for k_on of 3.3 × 10⁵ sec^–1 M^–1. The [Ac-Tyr¹, N⁴-proxyl,-D-Trp³²]-NPY antagonist ligand had a value of K_D equal to 1.35 × 10^–7 M and k_off was 1.7 × 10^–4 sec^–1 leading to a value for k_on of 1.2 × 10³ sec^–1 M^–1. The difference in the k_on rates of two orders of magnitude is interpreted as demonstrating the N-acetyl-N⁴ proxyl-D-Trp³²-NPY ligand binding transition state to be of higher energy then for the unmodified NPY amino acid sequence.     

Continuous production of glucoamylase by immobilized growing cells ofAureobasidium pullulans

Summary Yeast-like cells ofAureobasidium pullulans were immobilized in Ca-alginate gel beads and employed for continuous production of glucoamylase in a fluidized-bed reactor (250 ml working volume). After an activation time of 48 h, to allow the in situ germination of the fungal blastospores, the reactor was operated continuously for over 150 h. A steady state enzyme concentration of 1.2–1.3 U ml^–1 of glucoamylase activity and an enzyme volumetric productivity of ca. 130 U ml^–1 h^–1 were obtained at a medium flow rate of 26 ml h^–1. Enzyme activity and volumetric productivity were influenced by fermentation conditions such as inoculum size and airflow rate.     

Lack of effect of thrombin on fibrin(ogen)-endothelial cell interaction

In the present study we investigate the fibrin(ogen)-endothelial cell binding and the effect of thrombin on the endothelial cells in relation to fibrin(ogen) binding capacity. Endothelial cell fibrinogen binding was concentration and time-dependent, reaching saturation at 1.4 M of added ligand. At equilibrium, the number of fibrinogen molecules bound per endothelial cell in the monolayer was 5.8±0.7×10⁶. When endothelial cells were activated by different concentrations of thrombin (0–0.1 NIH units ml^–1), no increase in fibrinogen binding capacity was observed at all the thrombin concentration tested. Whereas disruption of endothelial cell monolayers was observed at thrombin concentrations higher than 0.05 NIH units ml^–1, no increase in the amount of fibrinogen bound was observed. Therefore, resting and thrombin-activated endothelial cells show the same fibrinogen binding capacity.The adhesion of endothelial cells in suspension on immobilized fibrinogen or fibrin was studied to ascertain whether the behavior of fibrin is similar to that of fibrinogen. The extent of endothelial cell attachment to immobilized fibrinogen and fibrin was similar (4275±130 cells cm^–2 for fibrinogen and 4350±235 cells cm^–2 for fibrin) and represent approximately 40% of the added endothelial cells. However, endothelial cell adhesion to immobilized fibrin was significantly faster than endothelial cell adhesion to immobilized fibrinogen. The maximum binding rate was 66±9 and 46±8 cells cm^–2 min^–1 for fibrin and fibrinogen, respectively. Therefore, the fibrinopeptides released by thrombin from fibrinogen induce qualitative changes which enhance the fibrin interaction with the endothelial cells.     

Effects of GNA and other mannose binding lectins on development and fecundity of the peach-potato aphid Myzus persicae

Three mannose-binding lectins were assayed in artificial diets for their toxic and growth-inhibitory effects on nymphal development of the peach-potato aphid Myzus persicae. The snowdrop (Galanthus nivalis) lectin GNA was the most toxic, with an induced nymphal mortality of 42% at 1500 g ml^–1 (30 M) and an IC₅₀ (50% growth inhibition) of 630 g ml^–1 (13 M). The daffodil (Narcissus pseudonarcissus) lectin NPA and a garlic (Allium sativum) lectin ASA induced no significant mortality in the range 10–1500 g ml^–1, but did result in growth inhibition of 59% (NPA) and 26% (ASA) at 1500 g ml^–1 (40 M for NPA, 63 M for ASA). All three lectins were responsible for a slight but significant growth stimulation when ingested at 10 g ml^–1, reaching +26%, +18% and +11% over the control values for the garlic lectin, the daffodil lectin and the snowdrop lectin, respectively. GNA, as well as the glucose/mannose binding lectin Concanavalin A, were also provided at sublethal doses throughout the life cycle of the aphids, and effects on adult performance were monitored. Adult survival was not significantly altered, but both lectins adversely affected total fecundity and the dynamics of reproduction, resulting in significant reduction in calculated r _ms (population intrinsic rate of natural increase) on lectin-containing diets. These effects are discussed in relation to the use of transgenic plants expressing these toxic lectins for potential control of aphid populations.     

Gene therapy for diabetic rats by electroporational transfer of naked plasmid with human pre-pro-insulin gene into skeletal muscle

A naked plasmid with human pre-pro-insulin gene was transferred into skeletal muscle of diabetic rats by electric pulses and gene expression was detected. Blood glucose concentration was decreased from 24 mmol l^–1 to 8.5 mmol l^–1. Circulating insulin-like protein was increased significantly to 15–20 U ml^–1, while that of the control group injected with the empty vector remained at less than 10 U ml^–1. The low blood glucose concentration lasted for more than two months. These studies indicate that electroporational transfer of plasmid with human pre-pro-insulin gene into skeletal muscle could be a potential method of gene therapy for human diabetes mellitus.