Protein micelles from lipoxygenase 3

Heat-induced conformational changes in lipoxygenase 3 were characterized by differential scanning calorimetry. The positions of the observed transitions were sensitive to the composition of the buffer. In particular, lipoxygenase 3 heated in carbonate buffer at pH 8.0 formed large soluble aggregates. Variable-temperature circular dichroism revealed that the formation of the aggregates was not accompanied by the unfolding of the C-terminal domain, which is composed primarily of alpha-helix. The aggregates were investigated using size exclusion chromatography, native polyacrylamide gel electrophoresis, dynamic light scattering, and electron microscopy. The data were consistent with the formation of roughly spherical particles with an average hydrodynamic radius of 26 nm and an approximate composite molecular weight of 10,000,000 Da. To account for the formation of soluble aggregates from lipoxygenase 3, we propose that hydrophobic amino acid residues are exposed by unfolding of the N-terminal beta-barrel domain of the protein resulting in the formation of protein micelles with a hydrophilic surface composed of the C-terminal domains.     

Selective interleukin-12 synthesis defect in 12/15-lipoxygenase-deficient macrophages associated with reduced atherosclerosis in a mouse model of familial hypercholesterolemia

Targeted gene disruption or overexpression of 12/15-lipoxygenase in mice on the genetic background of apolipoprotein E or low density lipoprotein-receptor (LDL-R) deficiency has implicated 12/15-lipoxygenase in atherogenesis. The data support indirectly a role for 12/15-lipoxygenase in the oxidative modification of low density lipoprotein. In this study we set out to explore other potential mechanisms for 12/15-lipoxygenase in atherosclerosis using apolipoprotein B mRNA editing catalytic polypeptide-1/LDL-R double-deficient mice, a model highly related to the human condition of familial hypercholesterolemia. 12/15-Lipoxygenase deficiency in this strain led to approximately 50% decrease in aortic lesions in male and female mice at 8 months on a chow diet in the absence of cholesterol differences. While studying 12/15-lipoxygenase-deficient macrophages in culture, we discovered a remarkable selective defect (75-90% decrease) in interleukin-12 production but not in tumor necrosis factor-alpha or nitric oxide release, in response to lipopolysaccharide in the presence or absence of interferon-gamma priming. The lipopolysaccharide/interferon-gamma response was associated with a 33-50% decrease in nuclear interferon consensus sequence-binding protein, which is consistent with interferon consensus sequence-binding protein containing protein complex-dependent regulation of the interleukin-12 p40 gene. The decrease in interleukin-12 production was recapitulated in vivo in mouse aortas of the triple knockout group and was reflected in a marked decrease in interferon-gamma expression. The data provide support for a novel mechanism linking the 12/15-lipoxygenase pathway to a known immunomodulatory Th1 cytokine in atherogenesis.     

Cutting edge: resistance to apoptosis and continuous proliferation of dendritic cells deficient for TNF receptor-1

The individual roles of the two TNFRs on dendritic cells (DC) are poorly understood. Investigating bone marrow-derived DC from TNFR-deficient mice, we found that cultures from TNFR1(-/-) mice continue to form proliferating clusters for 6-9 mo. In contrast, DC derived from wild-type, TNFR2(-/-), or TNFR1/2(-/-) mice survived for only 3-4 wk. DC obtained from these TNFR1(-/-) long term cultures (LTC) mice show an unusual mixed immature/mature phenotype. The continuous proliferation of the LTC is GM-CSF dependent and correlates with decreased protein levels of the cyclin-dependent kinase inhibitors p27(KIP1) and p21(CIP1). Prolonged survival of TNFR1(-/-) DC appears to be independent from NF-kappaB and Bcl-2 pathways and is rather enabled by the down-regulation of CD95, resulting in the resistance to CD95 ligand-induced apoptosis. These data point to proapoptotic signals mediated via TNFR1 and antiapoptotic signals mediated via TNFR2 in DC.     

Courtship role reversal and deceptive signals in the long-tailed dance fly, Rhamphomyia longicauda

We examined the function of secondary sexual characters in the role-reversed, lekking behaviour of female long-tailed dance flies, Rhamphomyia longicauda Loew (Empididae), to test the hypothesis that the degree of abdominal distention is an honest female signal about the state of egg development. Female Rhamphomyia cannot hunt for prey and they receive all of their protein from males by exchanging copulations for nuptial prey gifts. Females compete for male gifts within leks that are organized for a brief period each evening before dark. Before hovering within leks, females swallow air, inflating expandable pouches on the pleural margins of the abdomen. The result is a large saucer-like abdomen which is further exaggerated by wrapping scaled pro-, meso- and metathoracic legs along its pleural margins. Male preference for an enlarged abdomen was confirmed by suspending plastic models of varying size from monofilament lines and recording which models attracted the most males. There was a positive relationship between egg development and abdominal distention in a related species, R. sociabilis (Williston), which lacks inflatable abdominal pouches. Multiple regression showed that in R. longicauda, abdominal inflation completely masks the state of egg development. We conclude that female R. longicauda deceive mate-seeking males with the unreliable message that eggs are nearing maturation in order to obtain a protein meal in exchange for copulation. Males that fail to identify a female bearing mature eggs risk near-certain cuckoldry and an increased probability that the female will die before oviposition. Copyright 2000 The Association for the Study of Animal Behaviour.     

Identification of CIP-1-associated regulator of cyclin B (CARB), a novel p21-binding protein acting in the G2 phase of the cell cycle

The cyclin-dependent kinase inhibitor p21(cip1) regulates cell cycle progression, DNA replication, and DNA repair by binding to specific cellular proteins through distinct amino- and carboxyl-terminal protein binding motifs. We have identified a novel human gene, CARB (CIP-1-associated regulator of cyclin B), whose product interacts with the p21 carboxyl terminus. Immunocytochemical analysis demonstrates that the CARB protein is perinuclear and predominantly associated with the centrosome and mitotic spindle poles. In addition, CARB is also able to associate with cyclin B1, a key regulator of mitosis. However, cyclin B1-CARB complex formation occurs preferentially in the absence of p21. Unexpectedly, overexpression of CARB is associated with a growth-inhibitory and ultimately lethal phenotype in p21(-/-) cells but not in p21(+/+) cells. These data identify a novel mechanism that may underlie the effects of p21 in the G(2)/M phases of the cell cycle.     

Supermolecular structure of photosystem II and location of the PsbS protein

This paper addresses the question of whether the PsbS protein of photosystem two (PS II) is located within the LHC II PS II supercomplex for which a three-dimensional structure has been obtained by cryoelectron microscopy and single particle analysis. The PsbS protein has recently been implicated as the site for non-photochemical quenching. Based both on immunoblotting analyses and structural considerations of an improved model of the spinach LHC II PS II supercomplex, we conclude that the PsbS protein is not located within the supercomplex. Analyses of other fractions resulting from the solubilization of PS Il-enriched membranes derived from spinach suggest that the PsbS protein is located in the LHC II-rich regions that interconnect the supercomplex within the membrane.     

Characterization of an integral protein of the brush border membrane mediating the transport of divalent metal ions

The transport of Fe(2+) and other divalent transition metal ions across the intestinal brush border membrane (BBM) was investigated using brush border membrane vesicles (BBMVs) as a model. This transport is an energy-independent, protein-mediated process. The divalent metal ion transporter of the BBM is a spanning protein, very likely a protein channel, that senses the phase transition of the BBM, as indicated by a break in the Arrhenius plot. The transporter has a broad substrate range that includes Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), and Zn(2+). Under physiological conditions the transport of divalent metal ions is proton-coupled, leading to the acidification of the internal cavity of BBMVs. The divalent metal ion transporter can be solubilized in excess detergent (30 mM diheptanoylphosphatidylcholine or 1% Triton X-100) and reconstituted into an artificial membrane system by detergent removal. The reconstituted membrane system showed metal ion transport characteristics similar to those of the original BBMVs. The properties of the protein described here closely resemble those of the proton-coupled divalent cation transporter (DCT1, Nramp2) described by, Nature. 388:482-488). We may conclude that a protein of the Nramp family is present in the BBM, facilitating the transport of Fe(2+) and other divalent transition metal ions.     

Telomerase expression restores dermal integrity to in vitro-aged fibroblasts in a reconstituted skin model

The lifespan of human fibroblasts and other primary cell strains can be extended by expression of the telomerase catalytic subunit (hTERT). Since replicative senescence is accompanied by substantial alterations in gene expression, we evaluated characteristics of in vitro-aged dermal fibroblast populations before and after immortalization with telomerase. The biological behavior of these populations was assessed by incorporation into reconstituted human skin. Reminiscent of skin in the elderly, we observed increased fragility and subepidermal blistering with increased passage number of dermal fibroblasts, but the expression of telomerase in late passage populations restored the normal nonblistering phenotype. DNA microarray analysis showed that senescent fibroblasts express reduced levels of collagen I and III, as well as increased levels of a series of markers associated with the destruction of dermal matrix and inflammatory processes, and that the expression of telomerase results in mRNA expression patterns that are substantially similar to early passage cells. Thus, telomerase activity not only confers replicative immortality to skin fibroblasts, but can also prevent or reverse the loss of biological function seen in senescent cell populations.