bluuebunny/crossref_metadata_2025 huggingface dataset:
Neurotensin induces mating in Saccharomyces cerevisiae cells that express human neurotensin receptor type 1 in place of the endogenous pheromone receptor
Authors: Leplatois Pascal , Josse Annick , Guillemot Marie , Febvre Martine , Vita Natalio , Ferrara Pascual , Loison Gérard
Published: March 2003

Heterologous expression of the human neurotensin receptor type I (hNT1‐R) has been achieved in the yeast Saccharomyces cerevisiae. Immunoanalysis of membranes prepared from cells expressing a c‐myc‐tagged version of hNT1‐R revealed multiple c‐myc cross‐reacting polypeptides of high molecular mass, suggesting that hNT1‐R was glycosylated in yeast. High‐affinity binding sites for 125I‐labeled‐[monoiodo‐Tyr3]neurotensin ([125I‐Tyr3]NT) were detected on hNT1‐R‐expressing cells with Kd and Bmax values of 3.2 nm and of 500 receptors per cell, respectively. Competition binding studies of neurotensin with SR142948 and SR48692, two nonpeptidic antagonists of hNT1‐R, indicated that the yeast‐produced recombinant receptor displayed the same pharmacological properties as hNT1‐R expressed in mammalian cells. Interestingly, neurotensin activated the pheromone pathway in hNT1‐R‐expressing cells in a dose‐dependent fashion, as revealed by a β‐galactosidase activity assay with a pheromone‐responsive Fus1::lacZ construct. Mutational inactivation of the SST2 and STE2 genes increased the level of β‐galactosidase activity in response to neurotensin by twofold. Recombinant hNT1‐R‐producing cells, which lacked the endogenous G‐protein‐coupled receptor for the alpha pheromone, mated with wild‐type MATα haploid cells in response to neurotensin, leading to bona fide diploid zygote formation. This is the first report of a mammalian receptor that can replace the endogenous pheromone receptor when produced in yeast, by signaling a fully effective, agonist‐induced, mating process.
Cloning, expression, purification and characterization of patatin, a novel phospholipase A
Authors: Hirschberg Hoang J. H. B. , Simons Jan‐Willem. F. A. , Dekker Niek , Egmond Maarten R.
Published: March 2003

Patatin is the major protein constituent of potato tubers and displays broad esterase activity. The native enzyme actually belongs to a highly homologous multigene family of vacuolar glycoproteins. From these, the patB2 patatin gene was selected and cloned into pUC19 without its signal sequence but with an N‐terminal histidine‐tag. This patatin was overexpressed under the control of the lac promotor in Escherichia coli strain DH5α. The protein was recovered as inclusion bodies, folded into its native state by solubilization in urea and purified to homogeneity. Starting with one gram of inclusion bodies, 19 mg of pure and active recombinant patatin was isolated, with even higher specific activity than the glycosylated wild‐type patatin purified from potato tubers. The purified enzyme showed esterolytic activity with p‐nitrophenylesters dissolved in Triton X‐100 micelles. The activity of patatin on p‐nitrophenylesters with different carbon chain lengths showed an optimum for p‐nitrophenylesters with 10 carbon atoms. Besides general esterolytic activity, the pure enzyme was found to display high phospholipase A activity in particular with the substrates 1,2‐dioctanoyl‐sn‐glycero‐3‐phosphocholine (diC8PCho) (127 U·mg−1) and 1,2‐dinonanoyl‐sn‐glycero‐3‐phosphocholine (diC9PCho) (109 U·mg−1).

Recently, the structure of human cytosolic PLA2 (cPLA2) was solved, showing a novel Ser‐Asp active site dyad [1]. Based on a partial sequence alignment of patatin with human cPLA2, we propose that patatin contains a similar active site dyad. To verify this assumption, conserved Ser, Asp and His residues in the family of patatins have been modified in patatin B2. Identification of active site residues was based on the observation of correctly folded but inactive variants. This led to the assignment of Ser54 and Asp192 as the active site serine and aspartate residues in patatin B2, respectively.
Factors determining the special redox properties of photosynthetic cytochrome b559
Authors: Roncel Mercedes , Ortega José M. , Losada Manuel
Published: March 2003

Factors controlling the redox properties of the two conventional forms of cytochrome b559, i.e. the unstable high‐potential form and the stable low‐potential form, have been further investigated using PSII‐enriched membranes from pea and spinach chloroplasts. The redox potential of the stable form of cytochrome b559 is pH independent both above pH 7.5 (E′m ≈ +110 mV) and below pH 6.0 (E′m ≈+203 mV), but it changes with a slope of 58 mV per pH unit between these two pH values. Thus, cytochrome b559 seems to have a single ionizing group influencing its redox potential, with a higher affinity for protons in the reduced form (pKred = 7.5) and a lower affinity in the oxidized form (pKox = 6.0); consequently, one unprotonated low‐potential form (LP) and one protonated intermediate‐potential form (IP). The redox potential of the high‐potential form (HP) is pH‐independent between pH 5.0 and 8.0, but its relative content (compared to the total amount of protein) decreases progressively above pH 7.0. This conversion to the stable LP form is interpreted as corresponding to the loss of a proton by one ionizing group, the protonation of which is essential for maintaining the unstable HP state. According to chemical modification experiments with diethylpyrocarbonate, one of the two histidine ligands of the heme seems to be the ionizing group responsible for the existence of both the protonated IP and HP forms. It is proposed that the difference between the IP and HP forms is due to the formation of an additional hydrogen bond between the protonated histidine and the protein in the HP state that stabilizes a special hydrophobic heme environment responsible for its high redox potential.
The thermal unfolding and domain structure of Na+/K+‐exchanging ATPase.
Authors: Grinberg Asya V. , Gevondyan Natalia M. , Grinberg Natalia V. , Grinberg Valerij Y.
Published: March 2003

The thermal unfolding and domain structure of Na+/K+‐ATPase from pig kidney were studied by high‐sensitivity differential scanning calorimetry (HS‐DSC). The excess heat capacity function of Na+/K+‐ATPase displays the unfolding of three cooperative domains with midpoint transition temperatures (Td) of 320.6, 327.5, 331.5 K, respectively. The domain with Td = 327.5 K was identified as corresponding to the β subunit, while two other domains belong to the α subunit. The thermal unfolding of the low‐temperature domain leads to large changes in the amplitude of the short‐circuit current, but has no effect on the ATP hydrolysing activity. Furthermore, dithiothreitol or 2‐mercaptoethanol treatment causes destruction of this domain, accompanied by significant disruption of the ion transporting function and a 25% loss of ATPase activity. The observed total unfolding enthalpy of the protein is rather low (≈ 12 J·g−1), suggesting that thermal denaturation of Na+/K+‐ATPase does not lead to complete unfolding of the entire molecule. Presumably, transmembrane segments retain most of their secondary structure upon thermal denaturation. The binding of physiological ligands results in a pronounced increase in the conformational stability of both enzyme subunits.
The biosynthesis of ovothiol A (N1‐methyl‐4‐mercaptohistidine)
Authors: Vogt Ryan N. , Spies Hendrik S. C. , Steenkamp Daniel J.
Published: March 2003

Crude extracts of Crithidia fasciculata catalyse the formation of 4‐mercapto‐l‐histidine, an intermediate in the biosynthesis of ovothiol A (N1‐methyl‐4‐mercaptohistidine), in the presence of histidine, cysteine, Fe2+ and pyridoxal phosphate. This activity was present in a 35–55% ammonium sulfate fraction that was shown to produce a transsulfuration intermediate in the absence of pyridoxal phosphate. The transsulfuration intermediate was isolated and identified as S‐(4′‐l‐histidyl)‐l‐cysteine sulfoxide. The synthase activity, partially purified by anion‐exchange chromatography, was shown to require oxygen and could be used to synthesize a number of isotopically labeled S‐(4′‐l‐histidyl)‐l‐cysteine sulfoxides. Sulfoxide lyase activity was partially resolved from the synthase by anion‐exchange chromatography. The phenylhydrazone of the product derived from the cysteine moiety of the sulfoxide coeluted with the phenylhydrazone of pyruvate on HPLC, but this assignment could not be confirmed by mass spectral analysis. S‐(4′‐[14C]l‐histidyl)‐[U‐13C3,15N]l‐cysteine sulfoxide was synthesized and converted to products of the lyase reaction in the presence of lactate dehydrogenase and NADH. The 13C‐labeled product was identified by 13C‐NMR spectroscopy as lactate and the primary product of the lyase reaction is therefore pyruvate. With S‐(4′[3H]l‐histidyl)‐[14C]l‐cysteine sulfoxide as the substrate [14C]lactate, [14C]cysteine and [3H]4‐mercaptohistidine could be detected as products of the lyase reaction, but the sum of the two thiol species exceeded the amount of sulfoxide substrate used. Evidence is presented that this anomaly was due to the utilization of sulfur from dithiothreitol for the formation of cysteine.
Structural determinants for ligand binding and catalysis of  triosephosphate isomerase
Authors: Kursula Inari , Partanen Sanna , Lambeir Anne‐Marie , Antonov Dmitry M. , Augustyns Koen , Wierenga Rik K.
Published: March 2003

The crystal structure of leishmania triosephosphate isomerase (TIM) complexed with 2‐(N‐formyl‐N‐hydroxy)‐aminoethyl phosphonate (IPP) highlights the importance of Asn11 for binding and catalysis. IPP is an analogue of the substrate d‐glyceraldehyde‐3‐phosphate, and it is observed to bind with its aldehyde oxygen in an oxyanion hole formed by ND2 of Asn11 and NE2 of His95. Comparison of the mode of binding of IPP and the transition state analogue phosphoglycolohydroxamate (PGH) suggests that the Glu167 side chain, as well as the triose part of the substrate, adopt different conformations as the catalysed reaction proceeds. Comparison of the TIM–IPP and the TIM–PGH structures with other liganded and unliganded structures also highlights the conformational flexibility of the ligand and the active site, as well as the conserved mode of ligand binding.
Domain–domain interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90 molecular chaperone
Authors: Nemoto Takayuki K. , Ono Toshio , Kobayakawa Takeshi , Tanaka Etsuko , Baba Tomomi T. , Tanaka Ki‐ichiro , Takagi Takashi , Gotoh Toshio
Published: March 2003

In the present study, we investigated the domain structure and domain–domain interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90. Limited proteolysis of recombinant HtpG, revealed three major tryptic sites, i.e. Arg7‐Gly8, Arg336‐Glu337 and Lys552‐Leu553, of which the latter two were located at the positions equivalent to the major cleavage sites of human HSP90α. A similar pattern was obtained by papain treatment under nondenaturing conditions but not under denaturing conditions. Thus, HtpG consists of three domains, i.e. Domain A, Met1–Arg336; domain B, Glu337–Lys552; and domain C, Leu553‐Ser624, as does HSP90. The domains of HtpG were expressed and their interactions were estimated on polyacrylamide gel electrophoresis under nondenaturing conditions. As a result, two kinds of domain–domain interactions were revealed: domain B interaction with domain A of the same polypeptide and domain C of one partner with domain B of the other in the dimer. Domain B could be structurally and functionally divided into two subdomains, the N‐terminal two‐thirds (subdomain BI) that interacted with domain A and the C‐terminal one‐third (subdomain BII) that interacted with domain C. The C‐terminal two‐thirds of domain A, i.e. Asp116–Arg336, were sufficient for the binding to domain B. We finally propose the domain organization of an HtpG dimer.
An extrusion strategy for the FeMo cofactor from nitrogenase.
Authors: Martin Lisandra L. , West Lee C. , Wu Bei
Published: March 2003

Iron–sulfur clusters are ubiquitous in biological systems, facilitating functions such as electron transfer (rubredoxins, ferredoxins, rieske centres), isomerization (aconitase) and small molecule activation such as dinitrogen reduction (nitrogenases). Of global importance and recently particular interest, is the iron–sulfur‐containing iron‐molybdenum cofactor (FeMoco) cluster that achieves the biological reduction of dinitrogen under mild conditions. This biologically unique cluster has proved difficult to investigate due to its extreme air sensitivity and the instability of the cluster's structural integrity, outside the protective protein matrix.

Here, we report a model iron–sulfur cluster (Roussins black salt (NH4)[Fe4S3(NO)7]) that has been used to achieve the first example of a metal cluster (guest) embedded within a pseudo‐protein, cyclodextrin (host). The product formed is supramolecular, that is, it contained no covalent bonds and was stabilized by predominantly entropy effects. Formation of a 1 : 1 complex between the host and the guest was established for the iron–sulfur cluster with either seven‐ or eight‐membered cyclodextrins (β‐ or γ‐cyclodextrin). A range of techniques was used to characterize the new complexes in both the solid and solution states. Electrospray mass spectra indicated the presence of parent ions of the host–guest complexes and electrochemistry was also used to define the redox behavior of the complexes. The iron–sulfur clusters were significantly more stable in the presence of the host cyclodextrin, as revealed by a negative shift for the reduction potential for the host–guest product. Using the β‐cyclodextrin as host, the reduction potential of the iron–sulfur cluster shifted more negative by 60 mV; the effect was even more dramatic for the larger γ‐cyclodextrin where the reduction potential for the cluster was shifted by 90 mV more negative than the ‘unbound’[Fe4S3(NO)7]– cluster.

This is the first example of a metal cluster, stabilized as a supramolecular complex in a ‘host’ environment outside of a covalently bonded protein matrix. Creating such stable environments for metal cofactors or clusters that otherwise spontaneously degrade or are catalytically inactive outside the protein matrix could have enormous practical value. Specific implications for the development of extrusion methods for FeMoco from nitrogenase are enormous, with previously difficult, high‐energy molecular transformations, such as dinitrogen to ammonia, now more realistically accessible.
Elevated lysosomal pH in neuronal ceroid lipofuscinoses (NCLs)
Authors: Holopainen Juha M. , Saarikoski Juhani , Kinnunen Paavo K. J. , Järvelä Irma
Published: March 2003

We report here the intracellular (pHi) and lysosomal pH in fibroblasts of six forms of neuronal ceroid lipofuscinoses (NCLs). Acid extrusion rate and pHi values were measured by the membrane‐permeant acetoxymethyl ester of the indicator dye, 2′,7′‐bis(carboxyethyl)‐5‐(and‐6)‐carboxy‐fluorescein (BCECF) and lysosomal pH by a spectrofluorometric assay utilizing a novel acidotropic probe, Lysosensor yellow/blue. Intracellular pH was normal in all NCLs. Elevated lysosomal pH was detected in all NCL forms except CLN2 and CLN8. Elevated pH most probably disturbs the catalytic activity of lysosomes and is one important factor in explaining accumulation of ceroid and lipofuscin‐like autofluorescent lipopigments characteristic of NCLs. Using the novel spectrofluorometric assay introduced in this study provides a fast and repeatable technique to measure intralysosomal pH from cell suspensions.
Synthesis, conformational analysis and biological activity of cyclic analogs of the octadecaneuropeptide ODN
Authors: Leprince Jérôme , Oulyadi Hassan , Vaudry David , Masmoudi Olfa , Gandolfo Pierrick , Patte Christine , Costentin Jean , Fauchère Jean‐Luc , Davoust Daniel , Vaudry Hubert , Tonon Marie‐Christine
Published: March 2003

The octadecaneuropeptide (ODN; QATVGDVNTDRPGLLDLK) and its C‐terminal octapeptide (OP; RPGLLDLK), which exert anxiogenic activity, have been previously shown to increase intracellular calcium concentration ([Ca2+]i) in cultured rat astrocytes through activation of a metabotropic receptor positively coupled to phospholipase C. It has also been found that the [d‐Leu5]OP analog possesses a weak antagonistic activity. The aim of the present study was to synthesize and characterize cyclic analogs of OP and [d‐Leu5]OP. On‐resin homodetic backbone cyclization of OP yielded an analog, cyclo1−8 OP, which was three times more potent and 1.4‐times more efficacious than OP to increase [Ca2+]i in cultured rat astrocytes. Cyclo1−8 OP also mimicked the effect of both OP and ODN on polyphosphoinositide turnover. Conversely, the cyclo1−8[d‐Leu5]OP analog was totally devoid of agonistic activity but suppressed the effect of OP and ODN on [Ca2+]i and phosphoinositide metabolism in astrocytes. The structure of these cyclic analogs has been determined by two‐dimensional 1H‐NMR and molecular dynamics. Cyclo1−8 OP exhibited a single conformation characterized by a γ turn comprising residues Pro2–Leu4 and a type III β turn encompassing residues Leu5–Lys8. Cyclo1−8[d‐Leu5]OP was present as two equimolar conformers resulting from cis/trans isomerization of the Arg–Pro peptide bond. These pharmacological and structural data should prove useful for the rational design of non peptidic ODN analogs.
NMR studies in aqueous solution fail to identify significant conformational differences between the monomeric forms of two Alzheimer peptides with widely different plaque‐competence, Aβ(1–40)ox and Aβ(1–42)ox
Authors: Riek Roland , Güntert Peter , Döbeli Heinz , Wipf Beat , Wüthrich Kurt
Published: March 2003

NMR studies of amyloid β‐peptides (Aβ) in aqueous solution provide a novel way in which to characterize the apparent Alzheimer’s disease‐related conformational polymorphism of Aβ. In the aqueous medium, neither of the polypeptides Aβ(1–40)ox or Aβ(1–42)ox (both of which contain a methionine sulfoxide at position 35) is folded into a globular structure, but they both deviate from random coil behavior by local conformational preferences of several short segments along the amino‐acid sequence. Differences between the solution structures of Aβ(1–40)ox and Aβ(1–42)ox are indicated only by decreased flexibility of the region from about residue 32 to the C‐terminus in Aβ(1–42)ox when compared to Aβ(1–40)ox. The lack of the observation of more extensive conformational differences between the two molecules is intriguing, considering that Aβ(1–42)ox in aqueous solution has much higher plaque‐competence than Aβ(1–40)ox.
Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi
Authors: Gueguen Yannick , Rolland Jean‐luc , Lecompte Odile , Azam Philippe , Le Romancer Gisèle , Flament Didier , Raffin Jean‐Paul , Dietrich Jacques
Published: March 2003

The complete genome sequence of the hyperthermophilic archaeon Pyrococcus abyssi revealed the presence of a family B DNA polymerase (Pol I) and a family D DNA polymerase (Pol II). To extend our knowledge about euryarchaeal DNA polymerases, we cloned the genes encoding these two enzymes and expressed them in Escherichia coli. The DNA polymerases (Pol I and Pol II) were purified to homogeneity and characterized. Pol I had a molecular mass of ≈ 90 kDa, as estimated by SDS/PAGE. The optimum pH and Mg2+ concentration of Pol I were 8.5–9.0 and 3 mm, respectively. Pol II is composed of two subunits that are encoded by two genes arranged in tandem on the P. abyssi genome. We cloned these genes and purified the Pol II DNA polymerase from an E. coli strain coexpressing the cloned genes. The optimum pH and Mg2+ concentration of Pol II were 6.5 and 15–20 mm, respectively. Both P. abyssi Pol I and Pol II have associated 3′→5′ exonuclease activity although the exonuclease motifs usually found in DNA polymerases are absent in the archaeal family D DNA polymerase sequences. Sequence analysis has revealed that the small subunit of family D DNA polymerase and the Mre11 nucleases belong to the calcineurin‐like phosphoesterase superfamily and that residues involved in catalysis and metal coordination in the Mre11 nuclease three‐dimensional structure are strictly conserved in both families. One hypothesis is that the phosphoesterase domain of the small subunit is responsible for the 3′→5′ exonuclease activity of family D DNA polymerase. These results increase our understanding of euryarchaeal DNA polymerases and are of importance to push forward the complete understanding of the DNA replication in P. abyssi.
ik3‐1/Cables is a substrate for cyclin‐dependent kinase 3 (cdk 3)
Authors: Yamochi Tadanori , Semba Kentaro , Tsuji Keitaro , Mizumoto Kiyohisa , Sato Hiroko , Matsuura Yoshiharu , Nishimoto Ikuo , Matsuoka Masaaki
Published: March 2003

p70ik3‐1 (a 70‐kDa protein) contains a cyclin box, and binds to p35cdk3in vivo and in vitro[Matsuoka, M., Matsuura, Y., Semba, K. & Nishimoto, I. (2000) Biochem. Biophys. Res. Commun.273, 442–447]. In spite of its structural similarity to cyclins, p70ik3‐1 does not activate cyclin‐dependent kinase 3 (cdk3)‐mediated phosphorylation of pRb, histone H1, or the C‐terminal domain of RNA polymerase II. Here, we report that Ser274 of p70ik3‐1 is phosphorylated by cdk2 or cdk3 bound to cyclin A and to cyclin E in vitro. We also found that in COS7 cells in which cyclin E and cdk3 were ectopically overexpressed, the phosphorylation level of Ser274 in coexpressed p70ik3‐1 is upregulated. We therefore conclude that p70ik3‐1 is a substrate for cdk3‐mediated phosphorylation.
Arginine 121 is a crucial residue for the specific cytotoxic activity of the ribotoxin α‐sarcin
Authors: Masip Manuel , Lacadena Javier , Mancheño José Miguel , Oñaderra Mercedes , Martínez‐Ruiz Antonio , Martínez del Pozo Álvaro , Gavilanes José G.
Published: March 2003

α‐Sarcin, a cyclizing ribonuclease secreted by the mould Aspergillus giganteus, is one of the best characterized members of a family of fungal ribotoxins. This protein induces apoptosis in tumour cells due to its highly specific activity on ribosomes. Fungal ribotoxins display a three‐dimensional protein fold similar to those of a larger group of microbial noncytotoxic RNases, represented by RNases T1 and U2. This similarity involves the three catalytic residues and also the Arg121 residue, whose counterpart in RNase T1, Arg77, is located in the vicinity of the substrate phosphate moiety although its potential functional role is not known. In this work, Arg121 of α‐sarcin has been replaced by Gln or Lys. These two mutations do not modify the conformation of the protein but abolish the ribosome‐inactivating activity of α‐sarcin. In addition, the loss of the positive charge at that position produces dramatic changes on the interaction of α‐sarcin with phospholipid membranes. It is concluded that Arg121 is a crucial residue for the characteristic cytotoxicity of α‐sarcin and presumably of the other fungal ribotoxins.
Palmitate decreases proton pumping of liver‐type cytochrome c oxidase
Authors: Lee Icksoo , Kadenbach Bernhard
Published: March 2003

The H+/e– stoichiometry of reconstituted cytochrome c oxidase from bovine kidney, containing subunit VIaL (liver type), is 0.5 under standard conditions but 1.0 on addition of 1% cardiolipin to the lipid mixture (asolectin). Low concentrations of palmitate (half‐maximal effect at 0.5 µm), but not laurate, myristate, stearate, oleate, 1‐hexadecanol, palmitoyl glycerol and palmitoyl CoA, decreased the H+/e– ratio in the presence of cardiolipin from 1.0 to 0.5, accompanied by an increase of coupled, but not of uncoupled respiration of proteoliposomes. Cardiolipin and palmitate did not influence the H+/e– stoichiometry and respiration of reconstituted cytochrome c oxidase from bovine heart, containing subunit VIaH (heart‐type). The H+/e– stoichiometry of the heart enzyme, however, is decreased from 1.0 to 0.5 by 5 mm intraliposomal ATP (instead of 5 mm ADP). It is assumed that palmitate binds to subunit VIaL. The partial uncoupling of proton pumping in cytochrome c oxidase is suggested to participate in mammalian thermogenesis.
Expression of cathepsins B, D and L in mouse corneas infected withPseudomonas aeruginosa
Authors: Dong Zhong , Katar Malkhan , Linebaugh Bruce E. , Sloane Bonnie F. , Berk Richard S.
Published: March 2003

C57BL/6J naïve and immunized mice were intracorneally infected withPseudomonas aeruginosa. Semi‐quantitative RT‐PCR was performed to detect cathepsin gene expression and the results were further confirmed by immunoblot analysis. The enzymatic activities of cathepsins B, D and L were measured by peptidase assays. Immunohistochemical staining was carried out to localize the expression of the cathepsins. Cathepsins B, D and L were detected in the normal cornea by RT‐PCR. A peptidase assay revealed activities of all three cathepsins under normal physiological conditions. In naïve mice, enzymatic activities of cathepsins B, D and L were all significantly enhanced when the corneas were infected withP. aeruginosaand the peak of the induction appeared around day 6 postinfection. Immunoblot analysis showed increased expression of cathepsins B, D and L. The infected corneal samples from immunized mice exhibited much lower induction of enzymatic activities compared to those from naïve mice. Immunohistochemistry showed that the expression of cathepsins in the normal cornea was restricted to the epithelial tissue while the induced expression of cathepsins was predominantly in the substantia propria. Our data revealed up‐regulated enzymatic activities of cathepsins B, D and L in the naïve corneas infected withP. aeruginosa, which correlated well with the inflammatory response. Immunization of mice againstP. aeruginosaattenuated the inducing effect on cathepsin expression caused by infection. The time sequence for induction of cathepsin proteins and enzymatic activities suggests a mechanism of host proteolytic degradation of the extracellular matrix resulting in corneal destruction afterP. aeruginosainfection.
Cytochrome c/cytochrome c oxidase interaction
Authors: Sampson Valerie , Alleyne Trevor
Published: March 2003

We investigated the interaction between cytochrome c oxidase and its substrate cytochrome c by catalyzing the covalent linkage of the two proteins to yield 1 : 1 covalent enzyme–substrate complexes under conditions of low ionic strength. In addition to the ‘traditional’ oxidized complex formed between oxidized cytochrome c and the oxidized enzyme we prepared complexes under steady‐state reducing conditions. Whereas for the ‘oxidized’ complex cytochrome c became bound exclusively to subunit II of the enzyme, for the ‘steady‐state’ complex cytochrome c became bound to subunit II and two low molecular mass subunits, most likely VIb and IV.

For both complexes we investigated: (a) the ability of the covalently bound cytochrome c to relay electrons into the enzyme, and (b) the ability of the covalently bound enzyme to catalyze the oxidation of unbound (exogenous) ferrocytochrome c. Steady‐state spectral analysis (400–630 nm) combined with stopped‐flow studies, confirmed that the bound cytochrome c mediated the efficient transfer of electrons from the reducing agent ascorbate to the enzyme. In the case of the latter, the half life for the ascorbate reduction of the bound cytochrome c and that for the subsequent transfer of electrons to haem a were both < 5 ms. In contrast the covalent complexes, when reduced, were found to be totally unreactive towards oxidized cytochrome c oxidase confirming that the previously observed reduction of haem a within the complexes occurred via intramolecular rather than intermolecular electron transfer.

Additionally, stopped‐flow analysis at 550 nm showed that haem a within both covalent complexes catalyzed the oxidation of exogenous ferrocytochrome c: The second order rate constant for the traditional complex was 0.55×106 m−1·s−1 while that for the steady‐state was 0.27×106 m−1·s−1. These values were approximately 25–50% of those observed for 1 : 1 electrostatic complexes of similar concentrations. These results combined with those of the ascorbate and the electrophoresis studies suggest that electrons are able to enter cytochrome c oxidase via two independent pathways. We propose that during enzyme turnover the enzyme cycles between two conformers, one with a substrate binding site at subunit II and the other along the interface of subunits II, IV and VIb. Structural analysis suggests that Glu112, Glu113, Glu114 and Asp125 of subunit IV and Glu40, Glu54, Glu78, Asp35, Asp49, Asp73 and Asp74 of subunit VIb are residues that might possibly be involved.
A family of expressed antifreeze protein genes from the moth, Choristoneura fumiferana
Authors: Doucet Daniel , Tyshenko Michael G. , Davies Peter L. , Walker Virginia K.
Published: March 2003

The freeze‐intolerant insect, Choristoneura fumiferana (spruce budworm), produces multiple antifreeze protein (AFP) isoforms for protection during the overwintering stage. We now report the cloning of AFP genes from insects; Afp‐Lu1 encodes a ≈ 9‐kDa AFP isoform, and Afp‐Iu1 encodes a ≈ 12‐kDa AFP isoform. Both CfAFP genes have similar structures with a single 3‐ to 3.6‐kb intron interrupting the coding region. The second exon of an additional CfAFP gene, 2.7a, encoding a new ≈ 9‐kDa isoform, was found 3.7 kb upstream of Afp‐Lu1 and demonstrates that some AFP family members are linked in tandem. This gene appears to encode an AFP with 68–76% identity to previously isolated CfAFPs. With its eight Cys residues necessary for disulfide bonding and five perfectly conserved ‘Thr button’ (Thr‐Xaa‐Thr) ice‐binding motifs, it can be modeled as a functional AFP. Southern blot analysis shows that there are ≈ 17 genes in this AFP family, with each of the isoforms represented by two to five gene copies. Transcript accumulation from Afp‐Lu1 and Afp‐Iu1 (or closely related genes) was maximal during the overwintering stage, while 2.7a transcripts were only detected in first instars, larvae that are normally found only in the summer. Contrary to expectations, this differential expression demonstrates that CfAFP gene family transcripts are primarily regulated during development, rather than by seasonally low temperatures.
Antimicrobial dendrimeric peptides
Authors: Tam James P. , Lu Yi‐An , Yang Jin‐Long
Published: March 2003

Dendrimeric peptides selective for microbial surfaces have been developed to achieve broad antimicrobial activity and low hemolytic activity to human erythrocytes. The dendrimeric core is an asymmetric lysine branching tethered with two to eight copies of a tetrapeptide (R4) or an octapeptide (R8). The R4 tetrapeptide (RLYR) contains a putative microbial surface recognition BHHB motif (B = basic, H = hydrophobic amino acid) found in protegrins and tachyplesins whereas the octapeptide R8 (RLYRKVYG) consists of an R4 and a degenerated R4 repeat. Antimicrobial assays against 10 organisms in high‐ and low‐salt conditions showed that the R4 and R8 monomers as well as their divalent dendrimers contain no to low activity. In contrast, the tetra‐ and octavalent R4 and R8 dendrimers are broadly active under either conditions, exhibiting relatively similar potency with minimal inhibition concentrations < 1 µm against both bacteria and fungi. Based on their size and charge similarities, the potency and activity spectrum of the tetravalent R4 dendrimer are comparable to protegrins and tachyplesins, a family of potent antimicrobials containing 17–19 residues. Compared with a series of linearly repeating R4 peptides, the R4 dendrimers show comparable antimicrobial potency, but are more aqueous soluble, more stable to proteolysis, less toxic to human cells and more easily synthesized chemically. These results suggest repeating peptides that cluster the charge and hydrophobic residues may represent a primitive form of microbial pattern‐recognition. Incorporating such knowledge in a dendrimeric design therefore presents an attractive approach for developing novel peptide antibiotics.
Novel protein phosphatases in yeast
Authors: Ariño Joaquín
Published: March 2003

During the last decade several novel yeast genes encoding proteins related to the PPP family of Ser/Thr protein phosphatases have been discovered and their functional characterization initiated. Most of these novel phosphatases display intriguing structural features and/or are involved in a number of important functions, such as cell cycle regulation, protein synthesis and maintenance of cellular integrity. While in some cases these genes appear to be restricted to fungi, in others similar proteins can be found in higher eukaryotes. This review will summarize the latest advances in our understanding about how these phosphatases are regulated and fulfil their functions in the yeast cell.
Transient contribution of mast cells to pulmonary eosinophilia but not to hyper‐responsiveness
Authors: Ogawa K. , Kaminuma O. , Kikkawa H. , Nakata A. , Asahina M. , Egan R. W. , Akiyama K. , Mori A.
Published: March 2003

Background We have recently demonstrated that the transfer of interleukin (IL)‐5‐producing CD4+ T cell clones into unprimed mice is sufficient for the development of eosinophilic inflammation in the bronchial mucosa upon antigen inhalation.

Objective The aim of this study was to elucidate the possible contribution of mast cells in eosinophilic inflammation and bronchial hyper‐responsiveness (BHR), and to discriminate between the roles of CD4+ T cells and mast cells.

Methods Mast cell‐deficient mice (WBB6F1‐W/Wv) and their congenic normal littermates (WBB6F1−+/+) were immunized with ovalbumin and challenged by inhalation with the relevant antigen.

Results Airway eosinophilia was induced with equivalent intensity in +/+ and W/Wv mice 6, 24, 96 and 216 h after antigen inhalation. In contrast, 48 h after antigen challenge, eosinophilic infiltration into the bronchial mucosa was significantly less pronounced in W/Wv mice than in +/+ mice. Anti‐CD4 monoclonal antibody (mAb), anti‐IL‐5 mAb, and cyclosporin A were administered next, demonstrating that the airway eosinophilia of W/Wv mice induced 48 h after antigen challenge was almost completely inhibited by each of these three treatments, but that of +/+ mice was significantly less susceptible. Bronchial responsiveness to acetylcholine was increased 48 h after antigen challenge and was not significantly different between +/+ and W/Wv mice. Administration of anti‐IL‐5 mAb completely inhibited the development of BHR in both +/+ and W/Wv mice.

Conclusion These results indicate that, in mice, mast cells do have a supplemental role in the development of pulmonary eosinophilia but not BHR. CD4+ T cells totally regulate these responses by producing IL‐5.
cDNA cloning and heterologous expression of the major allergens from peach and apple belonging to the lipid‐transfer protein family
Authors: Diaz‐Perales A. , Garcia‐Casado G. , Sanchez‐Monge R. , Garcia‐Selles F. J. , Barber D. , Salcedo G.
Published: March 2003

Background Lipid‐transfer proteins (LTPs) have been identified as major allergens of Rosaceae fruits in populations living in areas virtually free of Fagales trees, such as several Mediterranean communities. Pru p 3 and Mal d 3, the allergens from peach and apple, respectively, have a main clinical relevance in these areas.

Objetive To isolate and characterize cDNAs for Pru p 3 and Mal d 3,and to produce recombinant Pru p 3 in the yeast Pichia pastoris.

Methods cDNAs for both allergens were isolated by polymerase chain reaction using non‐degenerated primers. Expression of Pru p 3 was performed in P. pastoris using the pPIC 9 vector. The recombinant product was purified by gel‐filtration chromatography followed by RP‐HPLC. Immunodetection and immunoblot inhibition assays were carried out with sera from peach‐allergic patients.

Results The cDNAs for both Pru p 3 and Mal d 3 showed a 273 open reading frame coding for the 91 amino acid mature polypeptides. The deduced amino acid sequences exhibited N‐terminal regions fully identical to those previously determined for the natural peach and apple allergens. Pru p 3 was expressed in P. pastoris at 20 mg/L of culture medium. The recombinant allergen showed the same N‐terminal sequence (plus a glutamic acid added for proper extracellular expression) and apparent molecular size as natural Pru p 3. Both the recombinant and natural forms of Pru p 3 displayed similar immunoglobulin (Ig)E‐binding capacity in immunodetection and immunoblot inhibition assays.

Conclusions Comparison of the complete primary structures of mature Pru p 3 and Mal d 3 deduced from their corresponding cDNA clones supports the close relationship between both allergens. Recombinant Pru p 3 binds IgE in vitro like its natural counterpart. Therefore, it can be a useful tool for specific diagnosis and structural studies.
OZ OT EBP 21C: Australian occupational therapy, evidence‐based practice and the 21st century
Authors: Cusick Anne
Published: March 2003

Occupational therapy in Australia is entering the 21st century as a dynamic and growing profession. The adoption of evidence‐based practice is an important feature to ensure the profession’s continued success. There are also other issues evident in Australian occupational therapy today that need to be considered to ensure the profession’s continued growth. These are: the terminology that is and could be used in occupational therapy; the possibility of ‘collateral damage’ to occupational therapy concepts and processes if deterministic elements of evidence‐based practice predominate; and the importance of making explicit our national position on occupational therapy core concepts and processes. Regarding the latter, the author proposes that this position is best described as ‘finding unity in diversity’. This position reflects the national heritage, character and social/health priorities facing Australian occupational therapy. The implications of these issues are explored.
Basal metabolic rate in migratory waders: intra‐individual, intraspecific, interspecific and seasonal variation
Authors: Kvist A. , Lindström Å.
Published: March 2003

Basal metabolic rates (BMR) were measured in 36 adult and 119 juvenile waders of 19 species on autumn migration in southern Sweden.

 In a comparison with literature data, it was found that juvenile BMR was generally lower than at the onset of migration in the Arctic and slightly higher than on African wintering grounds.

 The seasonal differences may reflect local physiological adaptations or possibly a gradual decline from high premigratory levels due to growth. Our data contradict the idea that BMR is high during migration as an adaptation to generally high levels of energy expenditure.

 The allometric exponent, scaling BMR to body mass, was significantly higher within individuals (1·19) and within species (1·82) than among species (0·62).

 The high intra‐individual exponent indicates that non‐fat tissues, with a high metabolic activity, are involved in the mass changes during migratory stopover.

 The high intraspecific exponent indicates that tissues with a high metabolic activity contributed disproportionately to variation in body mass among individuals or that larger individuals had elevated mass specific metabolic rates of some tissues.
Dehydration tolerance and water vapour absorption in two species of soil‐dwelling Collembola by accumulation of sugars and polyols
Authors: Holmstrup M. , Sjursen H. , Ravn H. , Bayley M.
Published: March 2003

Physiological and biochemical responses to desiccation were studied in two species of hygrophilic Collembola,Folsomia fimetariaL. andProtaphorura armataTullberg. The temporal changes in water content, body fluid osmotic pressure, and accumulation of sugars and polyols during desiccation stress, equivalent to severe soil drought are reported.

 Both species initially lost about 50% of their initial content of osmotically active water. Within 7 daysF. fimetariahad re‐established their preliminary water content by absorbing water vapour from the atmosphere.Protaphorura armataalso significantly increased their water content over the following 10 days.

 Both species were able to resume their hyperosmotic status relative to the desiccating environment they were placed in. The accumulation of myo‐inositol and two other unidentified compounds (F. fimetaria) and trehalose (P. armata) made an important contribution to this phenomenon.

 These results suggest that water vapour absorption by accumulation of sugars and polyols may be widespread in soil‐dwelling Collembola.
Density‐dependent foraging effort of Deer Mice (Peromyscus maniculatus)
Authors: Davidson D. L. , Morris D. W.
Published: March 2003

Little is known about how population density affects the foraging behaviour of individuals. Simple models are developed to predict the net effect of density on the quitting‐harvest rates of optimal foragers. The theory was tested with experiments that measured the foraging behaviour of free‐ranging Deer Mice under control and reduced densities.

 An increased density of conspecifics may (a) reduce the costs of foraging by increasing competition for resources (reduces the energetic state of the forager; competition hypothesis) or (b) increase the costs of foraging by increasing the value of time spent on social activities (social benefits hypothesis).

 A reduction in the costs of foraging caused by competition will reduce the quitting‐harvest rate of an optimal forager, whereas an increase in the value of alternative activities will increase the quitting‐harvest rate. Both hypotheses predict a reduction in optimal foraging time with increased density.

 The hypothesis that applies to Deer Mice (Peromyscus maniculatus, Wagner) was assessed by measuring their foraging activity and quitting‐harvest rates at control and reduced population densities on four study plots located in boreal forest in north‐western Ontario, Canada.

 Deer Mice increased theirper capitaforaging activity and their quitting‐harvest rates when population densities were reduced. The results confirm the very important role of competition in the behaviour of optimal foragers.
Gape size allometry in pike reflects variation between lakes in prey availability and relative body depth
Authors: MAGNHAGEN C. , HEIBO E.
Published: March 2003

The gape size of the predator and the body depth of its prey are the main factors deciding whether a gape‐limited piscivore can ingest a potential prey fish. The connection between morphological variation in pike and its prey was studied in five lakes with different proportions of pike, perch and roach.

At low prey abundance the prey fish are larger than in lakes with a higher prey density. In contrast, the pike are smaller but have relatively high gapes in the lake with the lowest prey availability, and larger but with smaller gapes at the highest prey density. There is a positive correlation with relative gape size of pike and relative body depth of prey among the five lakes.

Using the body depth in perch and roach and the gape size in pike, the proportion of the prey populations that could be eaten by the pike was calculated. In the low prey density lakes the pike could ingest a smaller proportion of the prey than in the high prey density lakes where most pike can eat all available prey sizes.

Competition for food in a situation where prey are big may cause a selection for a higher gape size. Our results indicate that this mechanism could be the reason that pike from different lakes vary in relative gape size.
Calcium oxalate crystals in leaves ofPancratiumsickenbergeri: constitutive or induced defence?
Authors: Ruiz N. , Ward D. , Saltz D.
Published: March 2003

We investigated the genotypic effects of selection by herbivory by the dorcas gazelle,Gazella dorcasLinnaeus 1758, on the production of calcium oxalate crystals in the lilyPancratium sickenbergeriC. et Barbey in the Negev Desert.

A completely crossed experimental design was used to assess the effects of soil calcium concentration and herbivory (simulated by clipping) on the production of calcium oxalate crystals in the leaves of plants from four source populations with different levels of herbivory.

 Soil calcium concentration did not have significant effects on the production of calcium oxalate crystals in the leaf. The number of crystals did not increase significantly with the clipping treatments.

 There was no significant difference among plants germinated from seed from populations with high or low herbivory in the production of calcium oxalate.

 There was a trade‐off between investment in defence versus investment in growth, suggesting a cost to defence. Experimentally clipped plants grew more than unclipped plants. Families from high‐herbivory areas had a stronger regrowth capacity in the absence of herbivory.

Our results indicate that crystal production by these plants is not a sink for excess calcium uptake from the soil. We conclude that in this species calcium oxalate crystal production is for defence, and that this defence is constitutive rather than inducible. The environmental unpredictability in this desert leaves plants with few cues to the onset of herbivory. Constitutive defence may be a more appropriate strategy than inducible defence in this environment, particularly when herbivory is high, and leaves are ephemeral.
Thermoregulatory ecology of a solitary bat, Myotis evotis, roosting in rock crevices
Authors: Chruszcz B. J. , Barclay R. M. R.
Published: March 2003

Most studies of the thermoregulatory ecology of bats have been limited to laboratory experiments or studies of individuals roosting in artificial structures.

 We investigated the interaction between thermoregulatory behaviour and roost choice in reproductive female Western Long‐Eared Bats, Myotis evotis (H. Allen), roosting solitarily in natural rock crevices. The study was conducted in the badlands of the South Saskatchewan River Valley, Alberta, Canada, during 1997 and 1998.

 Individuals used torpor every day and the amount of time spent in torpor was primarily influenced by the amount of time available to do so. Minimum body temperature was influenced by ambient temperature, although the form of this relationship differed between pregnant and lactating females. Pregnant females used deep torpor more frequently than lactating females.

 All individuals roosted in rock crevices but pregnant and lactating females chose roosts that were different in structure and thermal characteristics. Pregnant females chose horizontal roosts that cooled at night but warmed quickly during the day, thus allowing passive rewarming from torpor. Lactating females chose vertical roosts that stayed warm at night when non‐volant pups were present, thereby minimizing thermoregulatory costs to the young.

 The behaviours observed are adaptive, but differ from those of other temperate‐zone insectivorous bats that have been studied in the past. This highlights the importance of studying free‐ranging animals living in natural habitat if we are to have an accurate view of thermoregulatory strategies and the importance of roost characteristics for roost‐site selection.