The carbohydrate structures of a mouse monoclonal IgG antibody OKT3

A mouse monoclonal antibody OKT3, of IgG2a isotype, was isolated from hybridoma culture fluid. Sugar analysis showed the presence of sialic acid, galactose, mannose, fucose, and N-acetylglucosamine, i.e. sugars typical for N-glycosidically linked carbohydrate chains. The absence of N-acetylgalactosamine revealed that O-glycosidically linked carbohydrates were not present. The purified antibody was reduced, alkylated, and separated into heavy and light chains, and all carbohydrates were shown to be associated with the heavy chains. The N-linked carbohydrate chains were isolated as alditols using strong alkaline-borohydride degradation and further fractionated on a concanavalin A-Sepharose column and high performance ion exchange chromatography with pulsed amperometric detection. Structural analysis was carried out on the isolated oligosaccharide alditols by chemical analyses, fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. Triantennary and biantenary types of structures were found. The triantennary structures were present as trisialo and tetrasialo forms without fucose; the tetrasialo forms were shown to contain a sequence of Neu5Ac alpha 2-3Gal beta 1-3[Neu5Ac alpha 2-6]GlcNAc beta 1- on one of the branches. The biantennary structures were present as completely sialylated nonfucosylated species and as asialo-, agalacto-, and partially fucosylated structures.     

Virus-receptor interaction in the adenovirus system I. Identification of virion attachment proteins of the HeLa cell plasma membrane.

Plasma membranes from HeLa cells were isolated in a two-phase polymer system. To compare the efficiency of attachment protein extraction, a normalized assay for the assessment of adenovirus type 2 (Ad2) receptor-active components interfering with the attachment of Ad2 to HeLa cells was developed. An optimized detergent extraction procedure, 0.5% Triton X-100, was used, and solubilized membrane proteins were radioisotope labeled in vitro. Proteins with affinity for Ad2 virions were quantified and identified in a sucrose gradient sedimentation assay and by affinity chromatography with cross-linked Ad2 virions immobilized to AH-Sepharose 4B. From virions recovered in the sucrose gradient system, one major membrane component of high affinity was identified with a polypeptide molecular weight of around 40,000. Glycosylated proteins isolated by wheat germ lectin chromatography with high affinity for immobilized virus particles were isolated, and two major components with apparent molecular weights of 40,000 and 42,000 were identified. We suggest that a glycosylated protein with high affinity for Ad2 virions and a polypeptide molecular weight of 40,000 to 42,000 is one component of the Ad2 attachment site on HeLa cells.     

The mechanism of action of Cu2+ on the frog skin.

The mechanism of action of Cu2+ when applied to the external side of the frog skin preparation was investigated. Cu2+ acts most probably on the external barrier of this preparation, since it increases the transport pool of Na+ proportionally to the increase in the short circuit current (Isc). Cu2+ does not open new routes for the Na+ entry since the stimulated Isc is still completely abolished by amiloride. The Isc dependence of Na+ concentration in the external medium is modified by copper, since the Km value increases in addition to changes in V. It is suggested that copper acts at the external barrier Na channels in a way similar to that proposed by Zeiske and Lindemann ((1974) Biochim. Biophys. Acta 352, 323--326) for benzoylimidazole-2 guanidine and benzoylthiazole-2 guanidine and by Dick and Lindemann ((1975) Pflügers Arch. ges. Physiol. 355, R72) for para-chloromercuribenzenosulfonate and para-chloromercuribenzoate.     

Structural studies on the O-specific side-chains of the cell-wall lipopolysaccharide from Escherichia coli O 75

The structure of the O-specific side-chains of the cell-wall lipopolysaccharide of Escherichia coli O 75 has been investigated, using methylation analysis and Smith degradation as the principal methods. The O-specific side-chain was found to be composed of a tetrasaccharide repeating-unit of the following structure:

     

Biogenesis of microsomal membrane glycoproteins in rat liver. I. Presence of glycoproteins in microsomes and cytosol

The glycoproteins of microsomes and cytosol were studied. Various washing procedures did not release the proteins from the microsomes, and immunological tests demonstrated that the sialoproteins are not serum components. Low concentrations of deoxycholate and incubation in 0.25 M sucrose solution liberated a small amount of microsomal sialoprotein and this fraction exhibited a high degree of labeling of protein-bound N-acetylneuraminic acid. A part of the glycoprotein fraction could not be solubilized, even with a high concentration of the detergent. Thoroughly perfused rat liver contained sialoproteins in the particle-free supernate. The level of sialoprotein present could not be due to contamination with serum or broken organelles. The high in vivo incorporation of [3H]glucosamine into protein-bound sialic acid of Golgi membranes and cytosol was paralleled by a delayed and lesser rate of incorporation into the rough and smooth microsomal membranes. This incorporation pattern suggests the possibility that the glycoproteins of cytosol and Golgi may later be incorporated into the membrane of the endoplasmic reticulum.     

Sex-specific occurrence of androgen receptors in rat brain.

The metabolism and binding of [1, 2, 6, 7-3H] testosterone in male and female rat brain has been studied in an attempt to find an explanation for the relative androgen unresponsiveness characterizing the female hypothalamo-pituitary axis involved in regulation of hepatic steroid metabolism. The most significant sex differences in the pattern of [3H] testosterone metabolites recovered from several brain regions (including pituitary, pineal gland, and hypothalamus) after intraperitoneal administration of [3H] testosterone were the predominance of testosterone and androstenedione in male brain compared to the quantitative importance of 5alpha-androstane-3alpha, 17beta-diol, 5alpha-androstane-3beta, 17beta-diol, epitestosterone, and dihydroepitestosterone in female brain. One possible explanation for the androgen unresponsiveness of female rats is, therefore, the faster metabolism of testosterone to inactive compounds in female brain. Experiments both in vivo and in vitro showed the presence of high affinity, low capacity binding sites for [3H] testosterone in male pituitary, pineal gland, and hypothalamus (Kd values in the region of 1 X 10(-10) to 1 X 10(-9) M and number of binding sites 1.0 to 1.4 X 10(-14) mol per mg of protein). The steroid - macromolecular complexes generally had a pI of 5.1, were excluded from Sephadex G-200, were heat-labile, and were sensitive to protease. Competition experiments indicated the following order of ligand affinities: testosterone is greater than 5alpha-dihydrotestosterone and estradiol is greater than androstenedione is greater than corticosterone. No steroid-binding proteins of similar nature were found in pituitary, pineal gland, or hypothalamus from female rats. On the basis of these results it is suggested that the androgen unresponsiveness of female rats referred to above relates to the absence of receptor protein for androgens in female rat brain. In support of this hypothesis, 28-day-old female rats, which are known to be affected by androgens with regard to liver enzyme activities, were shown to contain receptor proteins for androgen in the brain. In conclusion, the relative androgen unresponsiveness of the female hypothalamo-pituitary axis is probably explained by the absence of receptor proteins for androgen in female hypothalamus and pituitary. The fast metabolism of testosterone in female rat brain also serves to decrease the availability of active androgen to potential receptor sites. It may be speculated that the presence of androgen receptors in male brain is the result of neonatal programming ("imprinting") by testicular androgen.     

Sperm adaptation in relation to salinity in three goby species

In externally fertilizing species, the gametes of both males and females are exposed to the influences of the environment into which they are released. Sperm are sensitive to abiotic factors such as salinity, but they are also affected by biotic factors such as sperm competition. In this study, the authors compared the performance of sperm of three goby species, the painted goby, Pomatoschistus pictus, the two-spotted goby, Pomatoschistus flavescens, and the sand goby, Pomatoschistus minutus. These species differ in their distributions, with painted goby having the narrowest salinity range and sand goby the widest. Moreover, data from paternity show that the two-spotted goby experiences the least sperm competition, whereas in the sand goby sperm competition is ubiquitous. The authors took sperm samples from dissected males and exposed them to high salinity water (31 PSU) representing the North Sea and low salinity water (6 PSU) representing the brackish Baltic Sea Proper. They then used computer-assisted sperm analysis to measure the proportion of motile sperm and sperm swimming speed 10 min and 20 h after sperm activation. The authors found that sperm performance depended on salinity, but there seemed to be no relationship to the species' geographical distribution in relation to salinity range. The species differed in the proportion of motile sperm, but there was no significant decrease in sperm motility during 20 h. The sand goby was the only species with motile sperm after 72 h.     

Courtship Sounds Advertise Species Identity and Male Quality in Sympatric Pomatoschistus spp. Gobies

Acoustic signals can encode crucial information about species identity and individual quality. We recorded and compared male courtship drum sounds of the sand goby Pomatoschistus minutus and the painted goby P. pictus and examined if they can function in species recognition within sympatric populations. We also examined which acoustic features are related to male quality and the factors that affect female courtship in the sand goby, to determine whether vocalisations potentially play a role in mate assessment. Drums produced by the painted goby showed significantly higher dominant frequencies, higher sound pulse repetition rates and longer intervals between sounds than those of the sand goby. In the sand goby, male quality was predicted by visual and acoustic courtship signals. Regression analyses showed that sound amplitude was a good predictor of male length, whereas the duration of nest behaviour and active calling rate (i.e. excluding silent periods) were good predictors of male condition factor and fat reserves respectively. In addition, the level of female courtship was predicted by male nest behaviour. The results suggest that the frequency and temporal patterns of sounds can encode species identity, whereas sound amplitude and calling activity reflects male size and fat reserves. Visual courtship duration (nest-related behaviour) also seems relevant to mate choice, since it reflects male condition and is related to female courtship. Our work suggests that acoustic communication can contribute to mate choice in the sand goby group, and invites further study.     

Near Infrared Optical Projection Tomography for Assessments of β-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen''s (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)²; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction² and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations³. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.     

Fungi Benefit from Two Decades of Increased Nutrient Availability in Tundra Heath Soil

If microbial degradation of carbon substrates in arctic soil is stimulated by climatic warming, this would be a significant positive feedback on global change. With data from a climate change experiment in Northern Sweden we show that warming and enhanced soil nutrient availability, which is a predicted long-term consequence of climatic warming and mimicked by fertilization, both increase soil microbial biomass. However, while fertilization increased the relative abundance of fungi, warming caused only a minimal shift in the microbial community composition based on the phospholipid fatty acid (PLFA) and neutral lipid fatty acid (NLFA) profiles. The function of the microbial community was also differently affected, as indicated by stable isotope probing of PLFA and NLFA. We demonstrate that two decades of fertilization have favored fungi relative to bacteria, and increased the turnover of complex organic compounds such as vanillin, while warming has had no such effects. Furthermore, the NLFA-to-PLFA ratio for ¹³C-incorporation from acetate increased in warmed plots but not in fertilized ones. Thus, fertilization cannot be used as a proxy for effects on warming in arctic tundra soils. Furthermore, the different functional responses suggest that the biomass increase found in both fertilized and warmed plots was mediated via different mechanisms.