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.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

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.