Functional analysis of a single chain chimeric alpha/beta-granulocyte-macrophage colony-stimulating factor receptor. Importance of a glutamate residue in the transmembrane region.

To analyze the function of each subunit of the receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF), GMR, we previously generated a single-chain chimeric receptor by fusion of the extracellular and transmembrane domain from the alpha-subunit (alpha-GMR) to the intracellular part of the beta-subunit (beta-GMR) introducing an additional glutamate residue at the fusion site (alpha/beta-GMR). We demonstrated the capacity of alpha/beta-GMR to bind GM-CSF with low affinity and to induce GM-CSF-dependent activation of tyrosine kinase activity and proliferation in transfected Ba/F3 cells. To further compare the functions of wild type and chimeric receptors, we now report that this alpha/beta-GMR is sufficient to mediate morphological changes, expression of alpha(4)- and beta(1)-integrin receptor subunits, and serine-phosphorylation of Akt kinase. To analyze the function of the glutamate residue at the fusion region of alpha/beta-GMR various point mutants changing this amino acid and its position were expressed in Ba/F3 cells. None of these mutants was capable of supporting GM-CSF-dependent proliferation; however, when beta-GMR was coexpressed, GM-CSF mediated short and long term proliferation. Interestingly, some mutants but not alpha/beta-GMR can induce proliferation in the presence of an anti-alpha-GMR antibody. These data demonstrate the significance of a glutamate residue in the transmembrane region of alpha/beta-GMR for ligand-induced receptor activation.     

SEASONAL VARIATION IN SEXUAL SELECTION IN THE MOTTLED SCULPIN

Seasonal variation in sexual and natural selection in male mottled sculpins (Cottus bairdi) can be evaluated by calculating selection differentials, which measure the magnitude of phenotypic change resulting from selection, and by calculating indices of the opportunity for selection, which indicate the potential for phenotypic selection in a given interval. Selection differentials are high at the beginning of the breeding season and decline throughout the breeding season. The magnitude and direction of selection differentials depend on when spawning occurs and are independent of the size or age of the females that spawn. Annual selection differentials due to differences in mating success (female choice) are nearly constant between years. Annual selection differentials associated with hatching success are variable. Opportunities for selection (I = fitness variance/[mean fitness]²) show clear seasonal patterns. They are highest at the beginning and at the end of the spawning season. However, this variation is dependent on the mean used to calculate I, and hence variation in I values does not indicate a significant change in the variance of male fitness.     

Distribution, purification and characterization of rat intestinal UDPgalactose: N-acetylglucosaminyl(beta 1----4)galactosyltransferase

Rat intestinal UDPgalactose: N-acetylglucosaminyl(beta 1----4)galactosyltransferase activity was studied as to its intestinal and villus-to-crypt distribution, and then purified and characterized. Rapid UDPgalactose hydrolysis was noted in the duodenum and jejunum; little to no breakdown was detected in the distal ileum, cecum and proximal colon. Product analysis suggested that UDPgalactose hydrolysis was due to nucleotide-sugar pyrophosphatase and galactose-1-phosphate phosphatase activities; ileum appeared to have little of the first activity and none of the latter. An aboral gradient of galactosyltransferase activity was noted, activity being 3-4-fold higher in the ileum, cecum and proximal colon. Total homogenate exogenous acceptor galactosyltransferase activities showed no villus-crypt differences but activity measured with intact isolated cells demonstrated higher activity with crypt cells; this was particularly evident in the ileum. Galactosyltransferase activity was purified from ileal-colonic mucosa. An over 4000-fold purification with 75 percent yield was achieved. Only one band of approx. 70-75 kDa was noted on sodium dodecyl sulfate polyacrylamide electrophoresis. As with other eukaryotic galactosyltransferase activities, there was an absolute requirement for Mn2+; the concentration required for half maximal activity was only 2.5 microM and higher concentrations did not inhibit. The Km for UDPgalactose was 30 microM.     

Characterization of envelope antigens of influenza A (H3N2) virus isolated during 1983-1985 epidemics and from sporadic cases of infection

Ten strains of influenza A (H3N2) virus isolated from an outbreak in 1983, and ten strains isolated in 1985 from sporadic cases of infection were included in the study. For characterization of envelope antigens were used the polyclonal and monoclonal antibodies tested in the reaction of haemagglutinin inhibition, neuraminidase inhibition, and by lectin test. The strains but slightly different in the tests with polyclonal antibodies could clearly be classified to 3-4 groups using 5 monoclonal antibodies to H antigen of A/Bangkok 1/79 and A/Philippines 2/82 strains. Strains from the 1983 epidemics represent a more homogeneous group of which only one of ten strains failed to react with monoclonals of the strains A/Bangkok and A/Philippines. Strains from sporadic cases of infection in 1985, except for two strains, did not react at all with the monoclonal discriminating A/Bangkok and A/Philippines. The other strains could be classified to three groups, i.e. whether they agreed with 4, 2 or none of the A/Philippines H antigen epitopes. Alterations of neuraminidase are less apparent, and cannot be defined by means of normal immune sera. With the use of monoclonal antibodies the strains under study do not react any more with the strains of 1968-1973 influenza virus; yet the monoclonals to A/Texas/77 strain still do recognize one or two epitopes of the 1983-1985 strains.     

The lectin neuraminidase inhibition test: a new method for the detection of antibodies to neuraminidase

Two methods for the detection of neuraminidase antibodies were compared. The lectin neuraminidase inhibition test (LNI-test) gave results comparable with those provided by the conventional neuraminidase inhibition test (NI-test). Reproducibility and repeatability were better with the LNI-test which used smaller amounts of materials, was less time consuming than the NI-test and was more sensitive.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Low species turnover of upland Amazonian birds in the absence of physical barriers

Aim

One of the oldest and most powerful ways for ecologists to explain distinct biological communities is to invoke underlying environmental differences. But in hyper-diverse systems, which often display high species richness and low species abundance, these sorts of community comparisons are especially challenging. The classic view for Amazonian birds posits that riverine barriers and habitat specialization determine local and regional community composition. We test the tacit, complementary assumption that similar bird communities should therefore permeate uniform habitat between major rivers, regardless of distance.

Location

Upland (terra firme) rainforests of central Amazonia.

Methods

We conducted intensive whole-community surveys of birds in three pairs of 100-ha plots, separated by 40–60 km. We then used dissimilarity indices, cluster analysis, and ordination to characterize differences among the six avian communities.

Results

In all, we detected 244 forest-dependent birds, with an average of 190 species (78%) per plot. Species turnover was negligible, no unique indicator species were found among plot pairs, and all documented species were already known from a complete inventory at one of the three sites.

Main Conclusions

Our study corroborates the classic biogeographical pattern and suggests that turnover contributes little to regional avian diversity within upland forests. Using a grain size of 100 ha, this implies that upland birds perceive the environment as uniform, at least over distances of ~60 km. Therefore, to maximize both local species richness and population persistence, our findings support the conservation of very large tracts of upland rainforest. Our analyses also revealed that the avifauna at Reserva Ducke, encroached by urban sprawl from the city of Manaus, shows the hallmarks of a disturbed community, with fewer vulnerable insectivores. This defaunation signals that even an enormous preserve (10 × 10 km) in lowland Amazonia is not insulated from anthropogenic degradation within the surrounding landscape.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Comparison of natural and synthetic prostaglandin E2 tablets in labour induction.

A multicentre, randomized, double-blind trial compared the efficacy and safety of and tolerance to natural and synthetically produced prostaglandin E2 tablets in the induction of labour in 202 women. The compounds were similarly effective, inducing labour in approximately 66% of patients. The total dose required and the interval between induction and delivery were similar in the two groups, as were the Apgar scores at 1 and 5 minutes and the incidence of maternal and fetal side effects.     

N-glycosylation is required for human CD2 immunoadhesion functions.

The T-lymphocyte glycoprotein receptor, CD2, mediates cell-cell adhesion by binding to the surface molecule CD58 (LFA-3) on many cell types including antigen presenting cells. Two domains comprise the CD2 extracellular segment, with all adhesion functions localized to the amino-terminal domain that contains a single N-glycosylation site at Asn65. We have defined an important role for the N-linked glycans attached to Asn65 of this domain in mediating CD2-CD58 interactions and also characterize its N-glycotype structure. Analysis of deglycosylated soluble recombinant CD2 as well as a mutant transmembrane CD2 molecule containing a single Asn65-Gln65 substitution demonstrates that neither deglycosylated CD2 nor the mutant CD2 transmembrane receptor binds CD58 or monoclonal antibodies directed at native CD2 adhesion domain epitopes. Electrospray ionization-mass spectrometry demonstrates that high mannose oligosaccharides ((Man)nGlcNAc2, n = 5-9) are the only N-glycotypes occupying Asn65 when soluble CD2 is expressed in Chinese hamster ovary cells. Based on a model of human CD2 secondary structure, we propose that N-glycosylation is required for stabilizing domain 1 in the human receptor. Thus, N-glycosylation is essential for human CD2 adhesion functions.