Functional characterization of the insulin-like growth factor I receptor on Jurkat T cells

Insulin-like growth factor I (IGF-I) has been shown to be important in the maintenance, development, and proliferation of various types of leukocytes, particularly T cells. Radio-receptor binding assays demonstrate that Jurkat T cells bind ¹²⁵I-IGF-I with an affinity of 1.77 nM (K_d) and express approximately 230 receptors/cell. Specificity studies show insulin also binds the IGF-I receptor with an affinity 20-fold lower than that of IGF-I. Interaction of IGF-I with its receptor on Jurkat T cells induces the phosphorylation of tyrosine kinase which is detectable by Western blotting. The 95,000 MW protein detected is equivalent to the molecular weight of the β chain of the IGF-I receptor described in other types of cells. These studies characterize the binding of IGF-I to its receptor on Jurkat T cells, demonstrate that IGF-I binding induces tyrosine phosphorylation, and support the hypothesis that IGF-I is important in the induction of T cell activation.     

Variation in lipid composition of some deep-sea fish (Teleostei: Oreosomatidae and Trachichthyidae)

The lipid, fatty acid and fatty alcohol compositions were determined for muscle samples from six species of deep-sea oreo collected from Australian waters; namely Neocyttus rhomboidalis, Neocyttus sp., Allocyttus verrucosus, Allocyttus niger, Pseudocyttus maculatus, and Oreosoma atlanticum. Neocyttus helgae, landed in North Atlantic waters, was also analysed. Similar analyses were also carried out on the muscle and swim bladder of the orange roughy Hoplostethus atlanticus from both Australian and North Atlantic waters. Orange roughy is currently a major commercial species in southern Australia and is a new-fishery in the North Atlantic; there are four species of oreo of increasing commercial significance in Australia due to orange roughy quota reductions. It is therefore necessary to determine if the oreo fishing industry is capable of supplementing the current orange roughy requirements with respect to muscle and oil demand. In the oreos, the mean lipid content ranged from 0.5 to 3% of wet weight, with a mixed lipid composition including wax ester, triacylglycerol, sterol and polar lipid. The ratio of the monounsaturated fatty alcohols 22:1 to 20:1 allowed samples from the two geographical regions to be distinguished. Total wax ester in muscle from North Atlantic male orange roughy was much higher than in Australian fish (27 vs. 8.5% wet weight, respectively); females from both locations contained similar amounts of wax ester (4.5 vs. 3.3%, respectively). Selected swim bladders from North Atlantic and Australian orange roughy show similar wax ester content (90 vs. 82%, respectively). The ratio of 22:1 to 20:1 fatty alcohols in orange roughy from the two regions was 0.5 (Australian) and 1.4 (North Atlantic). Indeed differences exist between oreos from the two locations, but not between orange roughy and this requires further investigation. With respect to the nutritional value, the oreos are more attractive than the orange roughy however lipid levels remain much lower compared with other popular species.     

Pain-reducing Properties of Sucrose in Human Newborns

To assess the characteristics of sucrose as a pain-reducingsubstance, crying in 72 newborn humans during and after bloodcollection via heel prick was determined. In the first studyinfants drank 2 ml of water or 2 ml of a 0.17-0.34- or 0.51-Msucrose solution 1 min prior to blood collection. In the secondexperiment, a delay of 30, 60, 90, 120 or 240 s was imposedbetween sucrose intake and the initiation of blood collection.The dose–response function for concentration was flat.The most effective time delay was 120 s. The effectiveness ofthe 2-min interval accords with previous findings of endogenousopioid release caused by sucrose taste. The flat dose–responsefunction extends findings in rats and humans that the calmingand pain-reducing effects of sucrose are not influenced by eitherconcentration or volume, suggesting that the transduction fromgustatory afferent to opioid-mediated efferent is of an on-offnature and not graded.     

Antibodies to tumor necrosis factor: a component of B cell immune responses with a role in tumor/host interaction

Infiltrating B lymphocytes are found within tumors, where their role and the antigens they recognize are poorly defined. After in vitro expansion of these cells, we were able to detect the production of antibodies to tumor necrosis factor (TNF) in 13 of 17 human tumors studied. These antibodies were detected by both enzyme-linked immunosorbent assay and by neutralization. Anti-TNF antibodies were not produced by resting peripheral blood B cells of normal subjects. However, anti-TNF antibodies were produced by B cells obtained from healthy individuals, after either in vivo or in vitro antigenic stimulation. This suggests that anti-TNF antibody production may constitute part of the overall B cell response to antigens. The intratumoral production of anti-TNF antibody may play a role in tumor/host interactions.This work was supported by grants from FAPESP (90/1844-4) and from the Share Foundation and the Concern Foundation     

A genomic clone containing a telomere array maps near the centromere of mouse Chromosome 6

A lambda clone of mouse DNA containing a short array of telomere hexamers has been localized by FISH to a region close to the centromere of Chromosome (Chr) 6. Amplification of DNA with primers flanking an SSR showed that most inbred strains carry one of two alleles, although five other alleles were found among the inbred strains and 11 other alleles were found in wild-derived mice. Analysis of the DNA from four Robertsonian translocations suggests that the amplified sequence is still present in these chromosomes. The finding of two fragments associated with the Sig mutant suggests that the clone lies within a congenic region created when the mutant, obtained in a (C3H x 101)F₁, was back-crossed to C57BL/6J. This region might include all or part of the centromere. Comparison of the segregation of the amplification product with the segregation of centromeric heterochromatin in an interspecies backcross, (C57BL/6 x M. spretus)F₁ x M. spretus, (BSS) shows 1/72 recombinants with the centromeric heterochromatin, while 1/62 recombinants occurred in a BSB backcross. Analysis of other loci at the proximal end of Chr 6 gives the combined map Hc6-0.73-D6Mit86-0.73-D6Rp2-2.2-D6Mitl-2.2-Wnt2-3.0-Cpa. Data from a third cross show that Cola2 lies between D6Mit82 and D6Rp2. The portion of the telomere array, Tel-rs3, that has been sequenced contains only 13/31 repeats of the consensus sequence. A variety of sequence changes from the consensus hexamer suggests that this array has been removed for a long time from evolutionary pressures to retain the TTAGGG sequence.     

Charactreization of polymorphism within the H-2M MHC class II loci

The products of the class II-like H2-M genes of the major histocompatibility complex are required for class II antigen processing. We sequenced H2-Ma and Mb from several mouse strains to determine whether these genes are polymophic, like H2-O. Both Mb loci appear to be transcribed and are distinct from each other. Mb1 and Mb2 differ by about 11% at the nucleotide level and are most dissimilar in their second exons (corresponding to the 1 domain). Relative to the published Mb 1 ^d haplotype sequence, the products of the b, g7, f, and k2 alleles of Mb 1 from Mus musculus domesticus and the separate mouse species Mus spretus differ by only one to four amino acids. The majority of the changes occured in the second exon of Mb 1, in contrast to HLA-DMB, the human orthologue. Little polymorphism was seen for Mb 2, and Ma was invariant in all strians tested. The similarity of the g7 allele to those from other haplotypes makes it unlikely that the M class II genes play a role in the autoimmune diabetes of NOD strain mice. The M genes are regulated in a manner similar to classical class II genes, in that they are upregulated by IFN-gd in mcrophages, and to a lesser extend by IL4 in B cells. When modeled on the crystal structure of the HLA-DR1 class II molecule, nearly all of the differences between M1 and M2 affect residues facing away from the putative peptide binding groove.     

Seasonal Changes in Scents and Responses to them in Meadow Voles: Evidence for the Co-evolution of Signals and Response Mechanisms

Relatively little is known about the mechanisms of communication during the non-breeding season in species that are seasonal breeders. Previous work with meadow voles, Microtus pennsylvanicus, housed under long photoperiods, has shown that they prefer the odors of opposite-sex conspecifics. In this paper, we investigated the effects of short photoperiod on preferences for sex-specific odors and the production of such odors. Short-photoperiod females preferred anogenital and fecal scents of other short-photoperiod females over those of males, but did not show sexual preferences for three other scents. Short-photoperiod males did not exhibit sexual preferences for any of the odors. Furthermore, scents from short-photoperiod voles did not elicit sex-specific preferences in long-photoperiod voles, and scents from long-photoperiod voles did not elicit preferences from short-photoperiod voles. These and previous results indicate that both the odors and responses to odors change seasonally and that long-photoperiod voles respond selectively to scents from long-photoperiod voles and short-photoperiod voles respond selectively to scents from short-photoperiod voles. Taken together, these results suggest the co-evolution of seasonal changes in scents and in perceptual or other response mechanisms.     

Soil respiration response to three years of elevated CO2 and N fertilization in ponderosa pine (Pinus ponderosa Doug. ex Laws.)

We measured growing season soil CO₂ evolution under elevated atmospheric [CO₂] and soil nitrogen (N) additions. Our objectives were to determine treatment effects, quantify seasonal variation, and compare two measurement techniques. Elevated [CO₂] treatments were applied in open-top chambers containing ponderosa pine (Pinus ponderosa L.) seedlings. N applications were made annually in early spring. The experimental design was a replicated factorial combination of CO₂ (ambient, + 175, and +350 L L^–1 CO₂) and N (0, 10, and 20 g m^–2 N as ammonium sulphate). Soils were irrigated to maintain soil moisture at > 25 percent. Soil CO₂ evolution was measured over diurnal periods (20–22 hours) in October 1992, and April, June, and October 1993 and 1994 using a flow-through, infrared gas analyzer measurement system and corresponding pCO₂ measurements were made with gas wells. Significantly higher soil CO₂ evolution was observed in the elevated CO₂ treatments; N effects were not significant. Averaged across all measurement periods, fluxes, were 4.8, 8.0, and 6.5 for ambient + 175 CO₂, and +350 CO₂ respectively).Treatment variation was linearly related to fungal occurrence as observed in minirhizotron tubes. Seasonal variation in soil CO₂ evolution was non-linearly related to soil temperature; i.e., fluxes increased up to approximately soil temperature (10cm soil depth) and decreased dramatically at temperatures > 18°C. These patterns indicate exceeding optimal temperatures for biological activity. The dynamic, flow-through measurement system was weakly correlated (r = 0.57; p < 0.0001; n = 56) with the pCO₂ measurement method.     

Activity of β-glucuronidase in Root Tips of Different Types of Transgenic Sugar Beet Plants

Expression of the -glucuronidase (GUS) reporter gene driven by the CaMV 35S, rolC, nos and mas promoters was assessed in the tips of 12 independent clones of transgenic sugar beet (Beta vulgaris) roots. Three questions were addressed: 1) expression pattern specific for a given promoter, 2) expression pattern variability, and 3) relationship between gene expression and cell differentiation. Characteristic patterns of tissue-specific expression were distinguished for each promoter. Striking differences, however, were found between some clones, bearing the same construct. Statistical analysis of the pattern variability proved that the variability is significantly lower within the construct than between constructs. rolC-GUS clones exhibited the lowest and CaMV 35S clones the highest pattern variability. Comparisons between the four promoters showed consistent GUS activity in areas playing a key role in tissue determination (the elongation zone) where cells switch from frequent mitosis and mostly isodiametrical growth, typical for the promeristem, to rapid elongation and differentiation. All of the clones were highly GUS-positive in the elongation zone of stele. Activity was commonly localised in the stele of the maturation zone for CaMV 35S, rolC and mas-GUS clones. CaMV 35S-GUS clones were highly active in the promeristem.     

Human Bak induces cell death in Schizosaccharomyces pombe with morphological changes similar to those with apoptosis in mammalian cells.

Apoptosis as a form of programmed cell death (PCD) in multicellular organisms is a well-established genetically controlled process that leads to elimination of unnecessary or damaged cells. Recently, PCD has also been described for unicellular organisms as a process for the socially advantageous regulation of cell survival. The human Bcl-2 family member Bak induces apoptosis in mammalian cells which is counteracted by the Bcl-x(L) protein. We show that Bak also kills the unicellular fission yeast Schizosaccharomyces pombe and that this is inhibited by coexpression of human Bcl-x(L). Moreover, the same critical BH3 domain of Bak that is required for induction of apoptosis in mammalian cells is also required for inducing death in yeast. This suggests that Bak kills mammalian and yeast cells by similar mechanisms. The phenotype of the Bak-induced death in yeast involves condensation and fragmentation of the chromatin as well as dissolution of the nuclear envelope, all of which are features of mammalian apoptosis. These data suggest that the evolutionarily conserved metazoan PCD pathway is also present in unicellular yeast.