Backbone dynamics of the human CC-chemokine eotaxin

Eotaxin is a CC chemokine with potent chemoattractant activity towards eosinophils. ¹⁵N NMR relaxation data have been used to characterize the backbone dynamics of recombinant human eotaxin. ¹⁵N longitudinal (R₁) and transverse (R₂) auto relaxation rates, heteronuclear ¹H-¹⁵N steady-state NOEs, and transverse cross-relaxation rates (_xy) were obtained at 30 °C for all resolved backbone secondary amide groups using ¹ H-detected two-dimensional NMR experiments. Ratios of transverse auto and cross relaxation rates were used to identify NH groups influenced by slow conformational rearrangement. Relaxation data were fit to the extended model free dynamics formalism, yielding parameters describing axially symmetric molecular rotational diffusion and the internal dynamics of each NH group. The molecular rotational correlation time (_m) is 5.09±0.02 ns, indicating that eotaxin exists predominantly as a monomer under the conditions of the NMR study. The ratio of diffusion rates about unique and perpendicular axes (D/D) is 0.81±0.02. Residues with large amplitudes of subnanosecond motion are clustered in the N-terminal region (residues 1–19), the C-terminus (residues 68–73) and the loop connecting the first two -strands (residues 30–37). N-terminal flexibility appears to be conserved throughout the chemokine family and may have implications for the mechanism of chemokine receptor activation. Residues exhibiting significant dynamics on the microsecond–millisecond time scale are located close to the two conserved disulfide bonds, suggesting that these motions may be coupled to disulfide bond isomerization.     

Role of Notch pathway in terminal follicle cell differentiation during Drosophila oogenesis

 During Drosophila oogenesis the body axes are determined by signaling between the oocyte and the somatic follicle cells that surround the egg chamber. A key event in the establishment of oocyte anterior-posterior polarity is the differential patterning of the follicle cell epithelium along the anterior-posterior axis. Both the Notch and epithelial growth factor (EGF) receptor pathways are required for this patterning. To understand how these pathways act in the process we have analyzed markers for anterior and posterior follicle cells accompanying constitutive activation of the EGF receptor, loss of Notch function, and ectopic expression of Delta. We find that a constitutively active EGF receptor can induce posterior fate in anterior but not in lateral follicle cells, showing that the EGF receptor pathway can act only on predetermined terminal cells. Furthermore, Notch function is required at both termini for appropriate expression of anterior and posterior markers, while loss of both the EGF receptor and Notch pathways mimic the Notch loss-of-function phenotype. Ectopic expression of the Notch ligand, Delta, disturbs EGF receptor dependent posterior follicle cell differentiation and anterior-posterior polarity of the oocyte. Our data are consistent with a model in which the Notch pathway is required for early follicle cell differentiation at both termini, but is then repressed at the posterior for proper determination of the posterior follicle cells by the EGF receptor pathway. Received: 5 November 1998 / Accepted: 14 December 1998     

Differential regulation of platelet-derived growth factor stimulated migration and proliferation in osteoblastic cells

Osteoblastic migration and proliferation in response to growth factors are essential for skeletal development, bone remodeling, and fracture repair, as well as pathologic processes, such as metastasis. We studied migration in response to platelet-derived growth factor (PDGF, 10 ng/ml) in a wounding model. PDGF stimulated a twofold increase in migration of osteoblastic MC3T3-E1 cells and murine calvarial osteoblasts over 24-48 h. PDGF also stimulated a tenfold increase in 3H-thymidine (3H-TdR) incorporation in MC3T3-E1 cells. Migration and DNA replication, as measured by BrdU incorporation, could be stimulated in the same cell. Blocking DNA replication with aphidicolin did not reduce the distance migrated. To examine the role of mitogen-activated protein (MAP) kinases in migration and proliferation, we used specific inhibitors of p38 MAP kinase, extracellular signal regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). For these signaling studies, proliferation was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) using flow cytometry. Inhibition of the p38 MAP kinase pathway by SB203580 and SB202190 blocked PDGF-stimulated migration but had no effect on proliferation. Inhibition of the ERK pathway by PD98059 and U0126 inhibited proliferation but did not inhibit migration. Inhibition of JNK activity by SP600125 inhibited both migration and proliferation. Hence, the stimulation of migration and proliferation by PDGF occurred by both overlapping and independent pathways. The JNK pathway was involved in both migration and proliferation, whereas the p38 pathway was predominantly involved in migration and the ERK pathway predominantly involved in proliferation.     

PKA-mediated phosphorylation of the beta1-adrenergic receptor promotes Gs/Gi switching

Recently, it has been shown that PKA-mediated phosphorylation of the β₂-adrenergic receptor (β₂-AR) by the cyclic AMP-dependent protein kinase (PKA) reduces its affinity for G_s and increases its affinity for G_i. Here we demonstrate that, like the β₂-AR, the β₁-AR is also capable of “switching” its coupling from G_s to G_i in a PKA-dependent manner. The β₁-AR is capable of activating adenylate cyclase via G_s, and can also activate the extracellular-regulated kinases, p44 and p42 (ERK1/2). In transfected CHO cells, the observed β₁-AR-mediated activation of ERK is both sensitive to pertussis toxin (PTX), indicating involvement of G_i/G_o, and to the PKA inhibitor, H-89. β₁-ARs with PKA phosphorylation sites mutated to alanines are unable to activate ERK. Mutating these same residues to aspartic acid, mimicking PKA phosphorylation, leads to a decrease in G_s-stimulated cAMP accumulation and an increase in PTX-sensitive ERK activation. These results strongly support the hypothesis that the β₁-AR, like the β₂-AR, can undergo PKA-dependent “G_s/G_i switching”.     

Comparison of methods to detect biosurfactant production by diverse microorganisms

Three methods to detect biosurfactant production, drop collapse, oil spreading, and blood agar lysis, were compared for their ease of use and reliability in relation to the ability of the cultures to reduce surface tension. The three methods were used to test for biosurfactant production in 205 environmental strains with different phylogenetic affiliations. Surface tension of select strains that gave conflicting results with the above three methods was also measured. Sixteen percent of the strains that lysed blood agar tested negative for biosurfactant production with the other two methods and had little reduction in surface tension (values above 60 mN/m). Thirty eight percent of the strains that did not lyse blood agar tested positive for biosurfactant production with the other two methods and had surface tension values as low as 35 mN/m. There was a very strong, negative, linear correlation between the diameter of clear zone obtained with the oil spreading technique and surface tension (rs = -0.959) and a weaker negative correlation between drop collapse method and surface tension (rs = -0.82), suggesting that the oil spreading technique better predicted biosurfactant production than the drop collapse method. The use of the drop collapse method as a primary method to detect biosurfactant producers, followed by the determination of the biosurfactant concentration using the oil spreading technique, constitutes a quick and easy protocol to screen and quantify biosurfactant production. The large number of false negatives and positives obtained with the blood agar lysis method and its poor correlation to surface tension (rs = -0.15) demonstrated that it is not a reliable method to detect biosurfactant production.     

Synthesis and biological activity of N-aryl-2-aminothiazoles: potent pan inhibitors of cyclin-dependent kinases

N-Aryl aminothiazoles 6-9 were prepared from 2-bromothiazole 5 and found to be CDK inhibitors. In cells they act as potent cytotoxic agents. Selectivity for CDK1, CDK2, and CDK4 was dependent of the nature of the N-aryl group and distinct from the CDK2 selective N-acyl analogues. The N-2-pyridyl analogues 7 and 19 showed pan CDK inhibitory activity. Elaborated analogues 19 and 23 exhibited anticancer activity in mice against P388 murine leukemia. The solid-state structure of 7 bound to CDK2 shows a similar binding mode to the N-acyl analogues.     

Differential accumulation and pigmenting ability of dietary carotenoids in colorful finches

Many animals develop bright red, orange, or yellow carotenoid pigmentation that they use to attract mates. Colorful carotenoid pigments are acquired from the diet and are either directly incorporated as integumentary colorants or metabolized into other forms before deposition. Because animals often obtain several different carotenoids from plant and animal food sources, it is possible that these pigments are accumulated at different levels in the body and may play unique roles in shaping the ultimate color expression of individuals. We studied patterns of carotenoid accumulation and integumentary pigmentation in two colorful finch species--the American goldfinch (Carduelis tristis) and the zebra finch (Taeniopygia guttata). Both species acquire two main hydroxycarotenoids, lutein and zeaxanthin, from their seed diet but transform these into a series of metabolites that are used as colorful pigments in the plumage (goldfinches only) and beak (both species). We conducted a series of carotenoid-supplementation experiments to investigate the relative extent to which lutein and zeaxanthin are accumulated in blood and increase carotenoid coloration in feathers and bare parts. First, we supplemented the diets of both species with either lutein or zeaxanthin and measured plasma pigment status, feather carotenoid concentration (goldfinches only), and integumentary color. Zeaxanthin-supplemented males grew more colorful feathers and beaks than lutein-supplemented males, and in goldfinches incorporated a different ratio of carotenoids in feathers (favoring the accumulation of canary xanthophyll B). We also fed goldfinches different concentrations of a standard lutein-zeaxanthin mix and found that at physiologically normal and high concentrations, birds circulated proportionally more zeaxanthin over lutein than occurred in the diet. Collectively, these results demonstrate that zeaxanthin is preferentially accumulated in the body and serves as a more potent substrate for pigmentation than lutein in these finches.     

Natal homing in juvenile loggerhead turtles (Caretta caretta)

Juvenile loggerhead turtles (Caretta caretta) from West Atlantic nesting beaches occupy oceanic (pelagic) habitats in the eastern Atlantic and Mediterranean, whereas larger juvenile turtles occupy shallow (neritic) habitats along the continental coastline of North America. Hence the switch from oceanic to neritic stage can involve a trans-oceanic migration. Several researchers have suggested that at the end of the oceanic phase, juveniles are homing to feeding habitats in the vicinity of their natal rookery. To test the hypothesis of juvenile homing behaviour, we surveyed 10 juvenile feeding zones across the eastern USA with mitochondrial DNA control region sequences (N = 1437) and compared these samples to potential source (nesting) populations in the Atlantic Ocean and Mediterranean Sea (N = 465). The results indicated a shallow, but significant, population structure of neritic juveniles (PhiST = 0.0088, P = 0.016), and haplotype frequency differences were significantly correlated between coastal feeding populations and adjacent nesting populations (Mantel test R2 = 0.52, P = 0.001). Mixed stock analyses (using a Bayesian algorithm) indicated that juveniles occurred at elevated frequency in the vicinity of their natal rookery. Hence, all lines of evidence supported the hypothesis of juvenile homing in loggerhead turtles. While not as precise as the homing of breeding adults, this behaviour nonetheless places juvenile turtles in the vicinity of their natal nesting colonies. Some of the coastal hazards that affect declining nesting populations may also affect the next generation of turtles feeding in nearby habitats.     

Promotion of peptide antimicrobial activity by fatty acid conjugation

Three peptides, YGAA[KKAAKAA](2) (AKK), KLFKRHLKWKII (SC4), and YG[AKAKAAKA](2) (KAK), were conjugated with lauric acid and tested for the effect on their structure, antibacterial activity, and eukaryotic cell toxicity. The conjugated AKK and SC4 peptides showed increased antimicrobial activity relative to unconjugated peptides, but the conjugated KAK peptide did not. The circular dichroism spectrum of AKK showed a significantly larger increase in its alpha-helical content in the conjugated form than peptide KAK in a solution containing phosphatidylethanolamine/phosphotidylglycerol vesicles, which mimics bacterial membranes. The KAK and AKK peptides and their corresponding fatty acid conjugates showed little change in their structure in the presence of phosphatidylcholine vesicles, which mimic the cell membrane of eukaryotic cells. The hemolytic activity of the KAK and AKK peptides and conjugates was low. However, the SC4 fatty acid conjugate showed a large increase in hemolytic activity and a corresponding increase in helical content in the presence of phosphatidylcholine vesicles. These results support the model of antimicrobial peptide hemolytic and antimicrobial activity being linked to changes in secondary structure as the peptides interact with lipid membranes. Fatty acid conjugation may improve the usefulness of peptides as antimicrobial agents by enhancing their ability to form secondary structures upon interacting with the bacterial membranes.     

Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila

We have used immunocytochemistry and cross-immunoprecipitation analysis to demonstrate that Megator (Bx34 antigen), a Tpr ortholog in Drosophila with an extended coiled-coil domain, colocalizes with the putative spindle matrix proteins Skeletor and Chromator during mitosis. Analysis of P-element mutations in the Megator locus showed that Megator is an essential protein. During interphase Megator is localized to the nuclear rim and occupies the intranuclear space surrounding the chromosomes. However, during mitosis Megator reorganizes and aligns together with Skeletor and Chromator into a fusiform spindle structure. The Megator metaphase spindle persists in the absence of microtubule spindles, strongly implying that the existence of the Megator-defined spindle does not require polymerized microtubules. Deletion construct analysis in S2 cells indicates that the COOH-terminal part of Megator without the coiled-coil region was sufficient for both nuclear as well as spindle localization. In contrast, the NH2-terminal coiled-coil region remains in the cytoplasm; however, we show that it is capable of assembling into spherical structures. On the basis of these findings we propose that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH2-terminal domain is responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex.