Shark skin: a function in feeding

Dermal denticles are unique tooth-like structures embedded in the skin of sharks and rays that protect them from predators and ectoparasites, reduce mechanical abrasion and possibly minimize swimming-induced drag. Here, we show that juvenile lesser spotted dogfish (Scyliorhinus canicula) also use this body armour to anchor food items near their tail so that bite-sized pieces can be torn away by rapid jaw and head movements. This scale-rasp behaviour is novel among fishes and suggests a new role for skin in the feeding ecology of sharks. Scale rasping may be important ecologically because it could function to increase the dietary breadth and growth potential of juveniles.     

High-precision,high-throughput stability determinations facilitated by robotics and a semiautomated titrating fluorometer

The use of statistical modeling to test hypotheses concerning the determinants of protein structure requires stability data (e.g., the free energy of denaturation in H(2)O, DeltaG(HOH)) from hundreds of protein mutants. Fluorescence-monitored chemical denaturation provides a convenient method for high-precision, high-throughput DeltaG(HOH) determination. For eglin c we find that a throughput of about 20 min per protein can be attained in a two-channel semiautomated titrating fluorometer. We find also that the use of robotics for protein purification and preparation of the solutions for chemical denaturation gives highly precise DeltaG(HOH) values in which the standard deviation of values from multiple preparations (+/-0.051 kcal/mol) differs very little from multiple measurements from a single preparation (+/-0.040 kcal/mol). Since the variance introduced into model fitting by DeltaG(HOH) increases as the square of measurement error, there is a premium on precision. In fact, the fraction of stability behavior explicable by otherwise perfect models goes from 98% to only 50% over the error range commonly reported for chemical denaturation measurements (0.1-0.6 kcal/mol). We have found that the precision of chemical denaturation DeltaG(HOH) measurements depends most heavily on the precision of the instrument used, followed by protein purity and the capacity to precisely prepare the solutions used for titrations.     

Palmitoylation of phospholipid scramblase 1 controls its distribution between nucleus and plasma membrane

Phospholipid scramblase 1 (PLSCR1) is a Ca(2+)-binding, endofacial plasma membrane protein thought to contribute to the transbilayer movement of phosphatidylserine and other membrane phospholipids that is observed upon influx of calcium into the cytosol. Expression of PLSCR1 is markedly induced by interferon and other cytokines, and PLSCR1-/- bone marrow cells exhibit defective myeloid proliferation and differentiation in response to stimulation by select growth factors, implying that PLSCR1 also functions in cytokine signaling or response pathways. PLSCR1 is multiply palmitoylated and partitions into membrane lipid raft domains. We have now identified the Cys-rich sequence (184)CCCPCC(189) in PLSCR1 as required for palmitoylation of the polypeptide. Mutation of these five cysteines abrogates PLSCR1 trafficking to the plasma membrane and results in virtually all of the expressed protein localizing to the nucleus. Consistent with this observation, cell treatment with the palmitoylation inhibitor, 2-bromo-palmitate, results in a marked redistribution of endogenous PLSCR1 from plasma membrane to nucleus. In a small percentage of untreated cells, predominantly nuclear localization of PLSCR1 is also observed. Furthermore, PLSCR1 is also found in the nucleus following its cytokine-induced expression. These data suggest that under the circumstance of rapid biosynthesis in response to gene induction by cytokines, PLSCR1 traffics into the nucleus, implying a potential nuclear function for this protein.     

Peptide-peptide interactions between human transferrin and transferrin-binding protein B from Moraxella catarrhalis

Transferrin-binding protein B (TbpB) is one component of a bipartite receptor in several gram-negative bacterial species that binds host transferrin and mediates the uptake of iron for growth. Transferrin and TbpB are both bilobed proteins, and the interaction between these proteins seems to involve similar lobe-lobe interactions. Synthetic overlapping peptide libraries representing the N lobe of TbpB from Moraxella catarrhalis were prepared and probed with labeled human transferrin. Transferrin-binding peptides were localized to six different regions of the TbpB N lobe, and reciprocal experiments identified six different regions of the C lobe of transferrin that bound TbpB. Truncations of the N lobe of TbpB that sequentially removed each transferrin-binding determinant were used to probe an overlapping peptide library of the C lobe of human transferrin. The removal of each TbpB N-lobe transferrin-binding determinant resulted in a loss of reactivity with peptides from the synthetic peptide library representing the C lobe of transferrin. Thus, individual peptide-peptide interactions between ligand and receptor were identified. A structural model of human transferrin was used to map surface regions capable of binding to TbpB.     

RANK ligand-induced elevation of cytosolic Ca2+ accelerates nuclear translocation of nuclear factor kappa B in osteoclasts

RANK ligand (RANKL) induces activation of NFkappaB, enhancing the formation, resorptive activity, and survival of osteoclasts. Ca(2+) transduces many signaling events, however, it is not known whether the actions of RANKL involve Ca(2+) signaling. We investigated the effects of RANKL on rat osteoclasts using microspectrofluorimetry and patch clamp. RANKL induced transient elevation of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) to maxima 220 nm above basal, resulting in activation of Ca(2+)-dependent K(+) current. RANKL elevated [Ca(2+)](i) in Ca(2+)-containing and Ca(2+)-free media, and responses were prevented by the phospholipase C inhibitor. Suppression of [Ca(2+)](i) elevation using the intracellular Ca(2+) chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) abolished the ability of RANKL to enhance osteoclast survival. Using immunofluorescence, NFkappaB was found predominantly in the cytosol of untreated osteoclasts. RANKL induced transient translocation of NFkappaB to the nuclei, which was maximal at 15 min. or BAPTA delayed nuclear translocation of NFkappaB. Delays were also observed upon inhibition of calcineurin or protein kinase C. We conclude that RANKL acts through phospholipase C to release Ca(2+) from intracellular stores, accelerating nuclear translocation of NFkappaB and promoting osteoclast survival. Such cross-talk between NFkappaB and Ca(2+) signaling provides a novel mechanism for the temporal regulation of gene expression in osteoclasts and other cell types.     

Intimal hyperplasia and wall shear in arterial bypass graft distal anastomoses: an in vivo model study

The observation of intimal hyperplasia at bypass graft anastomoses has suggested a potential interaction between local hemodynamics and vascular wall response. Wall shear has been particularly implicated because of its known effects upon the endothelium of normal vessels and, thus, was examined as to its possible role in the development of intimal hyperplasia in arterial bypass graft distal anastomoses. Tapered (4-7 mm I.D.) e-PTFE synthetic grafts 6 cm long were placed as bilateral carotid artery bypasses in six adult, mongrel dogs weighing between 25 and 30 kg with distal anastomotic graft-to-artery diameter ratios (DR) of either 1.0 or 1.5. Immediately following implantation, simultaneous axial velocity measurements were made in the toe and artery floor regions in the plane of the anastomosis at radial increments of 0.35 mm, 0.70 mm, and 1.05 mm using a specially designed 20 MHz triple crystal ultrasonic wall shear rate transducer Mean, peak, and pulse amplitude wall shear rates (WSRs), their absolute values, the spatial and temporal wall shear stress gradients (WSSG), and the oscillatory shear index (OSI) were computed from these velocity measurements. All grafts were harvested after 12 weeks implantation and measurements of the degree of intimal hyperplasia (IH) were made along the toe region and the artery floor of the host artery in 1 mm increments. While some IH occurred along the toe region (8.35+/-23.1 microm) and was significantly different between DR groups (p<0.003), the greatest amount occurred along the artery floor (81.6+/-106.5 microm, mean +/- S.D.) (p < 0.001) although no significant differences were found between DR groups. Linear regressions were performed on the paired IH and mean, peak, and pulse amplitude WSR data as well as the absolute mean, peak, and pulse amplitude WSR data from all grafts. The mean and absolute mean WSRs showed a modest correlation with IH (r = -0.406 and -0.370, respectively) with further improvements seen (r = -0.482 and -0.445, respectively) when using an exponential relationship. The overall best correlation was seen against an exponential function of the OSI (r = 0.600). Although these correlation coefficients were not high, they were found to be statistically significant as evidenced by the large F-statistic obtained. Finally, it was observed that over 75 percent of the IH occurred at or below a mean WSR value of 100 s(-1) while approximately 92 percent of the IH occurred at or below a mean WSR equal to one-half that of the native artery. Therefore, while not being the only factor involved, wall shear (and in particular, oscillators wall shear) appears to provide a stimulus for the development of anastomotic intimal hyperplasia.     

Effect of NOS inhibition on central response to atrial distension during pregnancy

Atrial distension increases c-fos expression in the paraventricular nucleus of virgin, but not pregnant, rats. We proposed that nitric oxide (NO), biosynthesis of which increases during pregnancy, blunts this reflex and that blocking NO biosynthesis would restore the response. Female rats were implanted with indwelling intracardiac balloons. On day 14 of pregnancy, osmotic minipumps containing either D- or N(G)-nitro-L-arginine methyl ester (L-NAME) (120 mg/2 ml at 10 microg/min) were implanted. On day 20, the rats were infused with saline (3 ml/h) with or without atrial balloon inflation (1 h). The brains were then processed for quantitation of c-fos expression. In the virgin rats, and in the pregnant rats treated with L-NAME, atrial distension significantly increased hypothalamic c-fos expression. In the pregnant animals treated with D-NAME, the response was greatly attenuated. NO had no effect on the increase in atrial receptor afferent discharge (single-fiber recordings) elicited by atrial distension. We conclude that, during pregnancy, NO attenuates central processing of the reflex response to atrial distension but does not alter the transducer properties of the volume receptors.     

Control of resistant pink bollworm (Pectinophora gossypiella) by transgenic cotton that produces Bacillus thuringiensis toxin Cry2Ab

Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 microg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 microg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 microg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.     

The role of the alpha2 chain in the stabilization of the collagen type I heterotrimer: a study of the type I homotrimer in oim mouse tissues

We have previously reported that the fragility of skin, tendon and bone from the oim mouse is related to a significant reduction in the intermolecular cross-linking. The oim mutation is unlikely to affect the efficacy of the lysyl oxidase, suggesting that the defect is in the molecule and fibre. We have therefore investigated the integrity of both the oim collagen molecules and the fibre by differential scanning calorimetry.The denaturation temperature of the oim molecule in solution and the fibre from tail tendon were found to be higher than the wild-type by 2.6deg.C and 1.9deg.C, respectively. With the loss of the alpha2 chain, the hydroxyproline content of the homotrimer is higher than the heterotrimer, which may account for the increase.There is a small decrease in the enthalpy of the oim fibres but it is not significant, suggesting that the amount of disorder of the triple-helical molecules and of the fibres is small and involves only a small part of the total bond energy holding the helical structure together. The difference in denaturation temperature of the skin collagen molecules (t(m)) and fibres (t(d)) is significantly lower for the oim tissues, 19.9deg.C against 23.1deg.C, indicating reduced molecular interactions and hence packing of the molecules in the fibre. Computation of the volume fraction of the water revealed that the interaxial separation of the oim fibres was indeed greater, increasing from 19.6A to 21.0A. This difference of 1.4A, equivalent to a C-C bond, would certainly decrease the ability of the telopeptide aldehyde to interact with the epsilon -amino group from an adjacent molecule and form a cross-link. We suggest, therefore, that the reduction of the cross-linking is due to increased water content of the fibre rather than a distortion of the molecular structure.The higher hydrophobicity of the alpha2 chain appears to play a role in the stabilisation of heterotrimeric type I collagen, possibly by increasing the hydrophobic interactions between the heterotrimeric molecules, thereby reducing the water content and increasing the binding of the molecules in the fibre.     

The characterisation of novel secreted Ly-6 proteins from rat urine by the combined use of two-dimensional gel electrophoresis, microbore high performance liquid chromatography and expressed sequence tag data

A proteomic study of rat urine was undertaken using two-dimensional gel electrophoresis, microbore high performance liquid chromatography, mass spectrometry and N-terminal sequencing. Five known urinary proteins were identified but two novel peptide fragments matched a large number of rat expressed sequence tags (ESTs) from a liver library. By combining protein chemical and nucleotide data, two 101-residue open reading frames with 90% amino acid identity were determined, rat urinary protein 1 (RUP-1) and RUP-2. The data established signal peptide removal and provided evidence for N-glycosylation. A third related sequence, rat spleen protein (RSP-1) was confirmed from EST searches. These three proteins have been submitted to SWISS-PROT as P81827, P81828 and Q9QXN2, respectively. A fourth novel homologue was found in porcine and bovine ESTs from embryo libraries. Alignment with known homologues showed conserved cysteine positions characteristic of a secreted subfamily of Ly-6 proteins. In two cases, antineoplastic urinary protein and caltrin, these homologues have unverified functional annotations. The RUP sequences showed high scoring matches to three unrelated rat mRNAs subsequently established to be chimeric. Two of these share extended sectional identity to RUP-1 but the third may represent another novel Ly-6 homologue. These chimeras have caused serious annotation errors in secondary databases.