Isolation and purification of serum and interfacial peptides of a trypsinolyzed β-lactoglobulin oil-in-water emulsion

Information on the conformation of proteins adsorbed to an oil–water interface is usually determined by following the time course of enzymatic hydrolysis of the protein in an oil-in-water emulsion. Unlike previous works reported in the literature, the research presented in this paper provides information on which peptides are actually in contact with the lipid bilayer (interfacial peptides) and those segments that project into the aqueous phase (serum peptides). In order to achieve this classification of peptides, we present a method to separate serum peptides from interfacial peptides by initial centrifugation steps followed by reversed-phase high-performance liquid chromatography. The effectiveness of the method was ascertained by performing proteolysis on β-lactoglobulin adsorbed to an oil-water interface in a soybean oil-water emulsion. It was found that more peptides are qualitatively and quantitatively found adsorbed to the oil–water interface as compared to peptides released into the serum.     

Repeated use of Bacillus subtilis cell walls for copper binding

Purified cell walls from Bacillus subtilis were repeatedly suspended in 5 mm CuCl₂ and, after removing unbound Cu, were suspended in 1% (v/v) HNO₃ to release bound Cu. The walls were then regenerated by washing in H₂O. After five cycles, copper binding actually increased slightly, probably due to enhanced exposure of binding sites in the walls. Thus bacterial walls may be used repeatedly for metal removal during bioremediation of heavy metal pollution.R.J.C. McLean is with the Department of Biology, Southwest Texas State University, 601 University Drive, San Marcos, TX 78666-4616, USA A.M. Campbell is with the Department of Chemical Engineering, Queen's University, Kingston, Ontario, K7L 3N6, Canada. P.T. Khu is with the Department of Mining Engineering, Queen's University, Kingston, Ontario, K7L 3N6, Canada. A.T. Persaud, L.E. Bickerton and D. Beauchemin are with the Department of Chemistry, Queen's University, Kingston, Ontario, K7L 3N6, Canada.     

Purification and characterisation of an odorant-binding protein from cow nasal tissue

Cow nasal tissue contains a protein which shows specific binding activity for 'green' smelling compounds such as 2-isobutyl-3-methoxypyrazine. This protein has now been purified using anion-exchange fast protein liquid chromatography. The protein has a relative molecular mass of 40 0000-44 000, s = 3.1 +/- 0.3 S, pI = 4.7 +/- 0.1 with an absorbance maximum at 278 nm, and consists of two subunits with an identical relative molecular mass of 19 000. It is localised in the soluble fraction of cells from the olfactory mucosa and respiratory mucosa from the middle part of the maxillary and nasal turbinates, and is absent from all other tissues tested.     

Effect of long-term application of animal manure on physical properties of three soils

Surface soil samples to 15 cm depth were taken from replicated plots in an ongoing long-term field experiment involving application of animal manure on three soils in Virginia. The sampled plots had received either no manure or the equivalent of 289,000 kg ha^–1 of manure as dry weight. The manure was applied annually at the beginning of each spring for 15 years from 1978 through 1992. The plots were cropped similarly since 1978. Soil textures were a fine sandy loam at Holland in the Atlantic Coastal Plain region, a silt loam at Blacksburg in the Appalachian region, and a clay loam at Orange in the Piedmont region of Virginia. The following measurements were made on subsamples: liquid and plastic limits, wet aggregate stability, aggregate size distribution, dispersible clay percentage, water retention at 0. 03, 0.1, 0.3, 0.5, 1.0, and 1.5 MPa tension, and modulus of rupture of moulded briquettes at a water content corresponding to 0.1 MPa tension. Organic matter content by the Walkley-Black method was significantly higher in the manure-treated soils at all three locations. Increases were 3% for the sandy loam and 25% for the silt loam and clay loam. From these values it was estimated that at least 95% of the total applied manure had been degraded over the 15 years. Results showed that the liquid and plastic limits for all three soils were higher (p<0.05) for the manure-treated samples. However, the differences in the limits were only 2 to 3%. The modulus of rupture values were lowered by addition of the animal manure. Decreases (p<0.05) occurred for the silt loam and clay loam samples. The wet aggregate stability increased and the dispersible clay decreased in the manure-treated soils. Increases (p<0.05) in wet aggregate stability occurred for the sandy loam and silt loam samples. Decreases (p<0.05) in dispersible clay were measured for the sandy loam and clay loam samples. Water retention was consistently, but only slightly, increased by manure addition. The increases, in the order of sample texture, were clay loam > sandy loam silt loam. Increases tended to be higher at the lower values of tension. Manure addition consistently increased the weight percentages of aggregates passing a given mesh size. Increases, in order of sample texture, were silt loam > clay loam > sandy loam. In their entirety, these results show that the manure produced measurable changes in the soil physical properties. The magnitude of the changes, in most cases, were small and depended on the soil texture. Given the high total amount of manure applied, the results indicate that manure-induced physical changes in the soil were small and evidently did not accumulate over time. Rapid microbial degradation of the manure could be responsible for the lack of marked changes in the soil physical properties.     

Influence of nicotine and caffeine on rat embryonic development

The influence on embryonic development of nicotine and caffeine at dose levels approximating human consumption was investigated in Sprague-Dawley rats. One group of animals received nicotine administered subcutaneously by an Alzet mini-osmotic pump from gestational day 6 through 12 (25 mg over 7 days; rate 149 micrograms/hr). Control animals received physiological saline in a similar manner. A second group received a single intravenous injection of caffeine (25 mg/kg) on gestational day 6. Control animals were treated with physiological saline. A further group received both nicotine and caffeine on gestational day 6 as described for the two previous groups. There were no significant differences among any of the groups with respect to maternal weight gain, litter size, embryolethality, fetal weight, or crown-rump length. The offspring of nicotine treated animals showed a significantly higher incidence of hydrocephaly when compared to the controls, but in the combined treatment group no malformed fetuses were observed. Light microscopic examination of maternal liver, kidney and placentas revealed changes in the hepatic sinusoids, glomeruli and intervillous spaces after nicotine and combined treatment. In addition, the decidua basalis was poorly developed compared to the controls. Chorionic villi and fetal kidney appeared normal in all groups. A coteratogenic effect is not evident from these findings.     

Expression and function of the extracellular calcium-sensing receptor in pancreatic beta-cells

The extracellular calcium-sensing receptor (CaR) was first identified in tissues involved in systemic Ca2+ homeostasis, where it acts to sense changes in circulating Ca2+. It has since been reported that the CaR is expressed in many tissues that are not associated with Ca2+ homeostasis, including the endocrine cells in pancreatic islets of Langerhans. In the present study we have used an insulin-secreting pancreatic beta-cell line (MIN6) to investigate the expression and function of CaR, using the calcimimetic A568, a CaR agonist that activates the CaR at physiological concentrations of extracellular Ca2+ ([Ca2+]o). Immunocytochemistry, Western blotting and RT-PCR confirmed the expression of CaR in MIN6 cells. CaR activation was associated with rapid and transient increases in [Ca2+]o, which were accompanied by the initiation of a marked but transient insulin secretory response. Stimulation of beta-cell secretory activity had no detectable effect on CaR mRNA levels, but CaR mRNA was markedly reduced by configuring MIN6 cells into islet- like structures. Our data are consistent with an important function for the beta-cell CaR in cell - cell communication within islets to co-ordinate insulin secretory responses.     

Simultaneous blockade of both the epidermal growth factor receptor and the insulin-like growth factor receptor signaling pathways in cancer cells with a fully human recombinant bispecific antibody

Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR) have been implicated in the tumorigenesis of a variety of human cancers. Effective tumor inhibition has been achieved both experimentally and clinically with a number of strategies that antagonize either receptor activity. Here we constructed and produced two fully human recombinant bispecific antibodies (BsAb) that target both EGFR and IGFR, using two neutralizing human antibodies originally isolated from a phage display library. The BsAb not only retained the antigen binding capacity of each of the parent antibodies, but also were capable of binding to both targets simultaneously as demonstrated by a cross-linking enzyme-linked immunosorbent assay. Furthermore, the BsAb effectively blocked both ligands, EGF and IGF, from binding to their respective receptors, and inhibited tumor cell proliferation as potently as a combination of both the parent antibodies. More importantly, the BsAb were able to completely block activation of several major signal transduction molecules, including Akt and p44/p42 MAP kinases, by both EGF and IGF, whereas each individual parent antibody was only effective in inhibiting those signal molecules activated by the relevant single growth factor. The BsAb molecules retained good antigen binding activity after incubation with mouse serum at 37 degrees C for up to 6 days. Taken together, our results underscore the benefits of simultaneous targeting multiple growth factor receptor pathways for more efficacious cancer treatment. This report describes the first time use of a recombinant BsAb for targeting two tumor-associated molecules on either a single or adjacent tumor cells for enhanced antitumor activity.     

Comparative immunogenicity in rhesus monkeys of DNA plasmid,recombinant vaccinia virus,and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene

Cellular immune responses, particularly those associated with CD3⁺ CD8⁺ cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5-gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4⁺ and CD8⁺ T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5-gag-induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5-gag. The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5-gag. These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting.