bluuebunny/crossref_metadata_2025_split huggingface dataset:
A comparison of kinetic and regulatory properties of the tetrameric and dimeric forms of wild‐type and Thr427→Pro mutant human phenylalanine hydroxylase
Authors: Bjørgo Elisa , de Carvalho Raquel Margarida Negrão , Flatmark Torgeir 
Published: March 2003

Recombinant human phenylalanine hydroxylase (hPAH, phenylalanine 4‐monooxygenase EC 1.14.16.1) is catalytically active both as a tetramer and a dimer [Knappskog, P.M., Flatmark, T., Aarden, J.M., Haavik, J. and Martínez, A. (1996) Eur. J. Biochem.242, 813–821]. In the present study we have further characterized the differences in kinetic and regulatory properties of the two oligomeric forms when expressed in Escherichia coli. The positive cooperativity of l‐Phe binding to the tetrameric form both in enzyme kinetic studies (h = 1.6) and intrinsic tryptophan fluorescence measurements (h = 2.3) was abolished in the dimer, which also revealed a catalytic efficiency (Vmax/[S]0.5) of only 35% of the tetramer. Whereas the catalytic activity of the tetramer was activated fivefold to sixfold by preincubation with l‐Phe, the dimer revealed only a 1.6‐fold activation. The crystal structure has identified a five‐residue flexible hinge region (Asp425–Gln429) that links the β‐strand Tβ2 (Ile421–Leu424) and the 24 residue amphipathic α‐helix Tα1 (Gln428–Lys452) at the C‐terminus which forms an antiparallel coiled‐coil structure in the center of the tetramer [Fusetti, F., Erlandsen, H., Flatmark, T. & Stevens, R.C. (1998) J. Biol. Chem.273, 16962–16967]. The potential role of this flexible hinge in the tetramerization and the conformational transition of wt‐hPAH on the cooperative binding of l‐Phe was examined by site‐specific mutagenesis. Substitution of Thr427 by a Pro (as in tyrosine hydroxylase) resulted in a mutant protein which was isolated mainly (about 95%) as a dimer. The isolated tetramer of T427P revealed no kinetic cooperativity of l‐Phe binding, the catalytic efficiency (Vmax/[S]0.5) was decreased to about 39% of the wild‐type tetramer and it was not activated by l‐Phe preincubation. The dimeric forms of T427P and wt‐hPAH revealed rather similar kinetic properties. The lack of kinetic cooperativity of the T427P tetramer was associated with a corresponding change in the binding isotherm for l‐Phe as studied by intrinsic tryptophan fluorescence measurements. Protein stability was also reduced both for the E. coli expressed and the in vitro synthesized mutant enzyme. Collectively, these results indicate that the positive cooperativity of l‐Phe binding to wt‐hPAH requires a tetrameric enzyme with a C‐terminal flexibel hinge region (Asp425–Gln429) which has a structural role in the formation of the enzyme tetramer. Furthermore, this hinge region represents a motif in the PAH structure that is involved in the conformational change transmitted through the protein on the cooperative binding of l‐Phe to tetrameric wt‐hPAH. This conclusion is further supported by studies on two disease (phenylketonuria)‐associated mutant forms.
Growth factor‐stimulated protein synthesis is inhibited by sodium orthovanadate
Authors: Viñals Francesc , McKenzie Fergus R. , Pouysségur Jacques 
Published: March 2003

The study of intracellular signaling pathways has been aided by the use of sodium orthovanadate, a cell‐permeable inhibitor of tyrosine phosphatases. However, long‐term addition of sodium orthovanadate is often cytotoxic. In this study we demonstrate that the growth factor‐mediated increase in the rate of protein synthesis was inhibited by sodium orthovanadate. This effect of sodium orthovanadate was dose‐dependent, with an IC50 of 40 µm and maximal inhibition obtained at 100 µm. As a consequence, the fetal bovine serum‐mediated induction of the immediate‐early genes, c‐Fos and MKP‐1, at the protein level was inhibited by orthovanadate. Orthovanadate’s ability to attenuate protein synthesis was partially reversible, and was no longer evident when the agent was added 6 h after addition of growth factor to cells. Analysis of several elements of signaling pathways which are known to regulate protein synthesis in a positive manner (p42/p44 MAPK, AKT and p70 S6K stimulation, and hyperphosphorylation of PHAS‐I) were not inhibited but rather were stimulated by orthovanadate. Thus, sodium orthovanadate is a potent inhibitor of growth factor‐stimulated protein synthesis independent of p42/p44 MAPK or PI3K‐p70 S6K activation.
Complete primary structure of rainbow trout type I collagen consisting of α1(I)α2(I)α3(I) heterotrimers
Authors: Saito Masataka , Takenouchi Yoshitaka , Kunisaki Naomichi , Kimura Shigeru 
Published: March 2003

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be α1(I)α2(I)α3(I) and [α1(I)]2α2(I), respectively. The occurrence of α3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP‐13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 °C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N‐terminal and/or a part of triple‐helical domains of proα(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple‐helical domain of each proα(I) chain had 338 uninterrupted Gly‐X‐Y triplets consisting of 1014 amino acids and was unique in its high content of Gly‐Gly doublets. In particular, the bonyfish‐specific α(I) chain, proα3(I) was characterized by the small number of Gly‐Pro‐Pro triplets, 19, and the large number of Gly‐Gly doublets, 38, in the triple‐helical domain, compared to 23 and 22, respectively, for proα1(I). The small number of Gly‐Pro‐Pro and the large number of Gly‐Gly in proα3(I) was assumed to partially loosen the triple‐helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proα3(I) had diverged from proα1(I). This study is the first report of the complete primary structure of fish type I procollagen.
Mutation of residues in the coenzyme binding pocket of Dopa decarboxylase
Authors: Bertoldi Mariarita , Castellani Silvia , Voltattorni Carla Borri 
Published: March 2003

Residues D271, H192, H302 and N300 of l‐3,4‐dihydroxyphenylalanine decarboxylase (DDC), a homodimeric pyridoxal 5′‐phosphate (PLP) enzyme, were mutated in order to acquire information on the catalytic mechanism. These residues are potential participants in catalysis because they belong to the common PLP‐binding structural motif of group I, II and III decarboxylases and other PLP enzymes, and because they are among the putative active‐site residues of structural modelled rat liver DDC. The spectroscopic features of the D271E, H192Q, H302Q and N300A mutants as well as their dissociation constants for PLP suggest that substitution of each of these residues causes alteration of the state of the bound coenzyme molecule and of the conformation of aromatic amino acids, possibly in the vicinity of the active site. This supports, but does not prove, the possibility that these residues are located in the coenzyme‐binding cleft. Interestingly, mutation of each residue generates an oxidative decarboxylase activity towards l‐3,4‐dihydroxyphenylalanine (l‐Dopa), not inherent in the wild‐type in aerobiosis, and reduces the nonoxidative decarboxylase activity of l‐Dopa from 3‐ to 390‐fold. The partition ratio between oxidative and nonoxidative decarboxylation ranges from 5.7 × 10−4 for N300A mutant to 946 × 10−4 for H302Q mutant. Unlike wild‐type enzyme, the mutants catalyse these two reactions to the same extent either in the presence or absence of O2. In addition, all four mutants exhibit an extremely low level of the oxidative deaminase activity towards serotonin with respect to wild‐type. All these findings demonstrate that although D271, H192, H302 and N300 are not essential for catalysis, mutation of these residues alters the nature of catalysis. A possible relationship among the integrity of the PLP cleft, the productive binding of O2 and the transition to a closed conformational state of DDC is discussed.
Possible involvement of the membrane‐bound form of peroxiredoxin 4 in acrosome formation during spermiogenesis of rats
Authors: Sasagawa Isoji , Matsuki Shingo , Suzuki Yasuhiro , Iuchi Yoshihito , Tohya Kazuo , Kimura Michio , Nakada Teruhiro , Fujii Junichi 
Published: March 2003

Peroxiredoxins are novel peroxidases that exhibit divergent biological functions. The fourth member, Prx4, is synthesized with a signal sequence and, after processing, secreted as a 27‐kDa form in most tissues. A 31‐kDa Prx4 which corresponds to the unprocessed, membrane‐bound form is found only in testis. This study was undertaken in order to investigate the role of the membrane‐bound Prx4 during spermiogenesis in rats. The Prx4 transcript was observed in testes at 14 days of age, the age before puberty, and its level increased only slightly during aging. Only the secretable form of Prx4 was present before 30 days of age, but the membrane‐bound Prx4 became detectable in adult testes, although sum of the two forms appeared to be the same after 21 days old. While weak immunoreactivity to the Prx4 antibody was detected in spermatogenic cells for all ages, a strong immunoreactivity was observed in the elongating spermatid and residual body of adult testis. Prx4 was present in the lumen of the endoplasmic reticulum, Golgi bodies, and the perinuclear space in young rat testes. However, the localization of Prx4 was restricted to membranes of the acrosomal vesicle of the elongated spermatid and was not detected in spermatozoon. Once spermiogenesis is accomplished, vesicles containing the membrane‐bound Prx4 were released into the residual body, along with residual membranous components. Thus the conversion of the soluble form to the membrane‐bound form may have a role in the acrosome formation during vesicular reorganization during spermiogenesis.
Cell surface human α‐L‐fucosidase
Authors: Cordero Oscar J. , Merino Ana , de la Cadena María Páez , Bugía Begoña , Nogueira Montserrat , Viñuela Juan E. , Martínez‐Zorzano Vicenta S. , de Carlos Alejandro , Rodríguez‐Berrocal F. Javier 
Published: March 2003

The acid α‐l‐fucosidase is usually found as a soluble component of lysosomes where fucoglycoconjugates are degraded. In the present investigation, we have demonstrated the existence of a cell surface protein with enzymatic α‐l‐fucosidase activity that crossreacts specifically with a rabbit anti‐(α‐l‐fucosidase) Ig. By different approaches, this α‐l‐fucosidase, which represents 10–20% of the total cellular fucosidase activity, was detected in all the tested human cells (hemopoietic, epithelial, mesenchymal). Two bands of ≈ 43–49 kDa were observed, although theoretical data support the possibility of having the same genetic origin that the known 50 to 55‐kDa Mrα‐l‐fucosidase. We speculate about an alternative traffic pathway for the plasma membrane α‐l‐fucosidase to work on the rapid turnover of glycoproteins.
Dimerization, stability and electrostatic properties of porcine β‐lactoglobulin
Authors: Ugolini Raffaella , Ragona Laura , Silletti Erika , Fogolari Federico , Visschers Ronald W. , Alting Arno C. , Molinari Henriette 
Published: March 2003

The study of homologous proteins belonging to the same family can provide a rationale for important molecular properties such as oligomer formation, folding mechanism and mode of binding. We report here a physico‐chemical characterization of porcine β‐lactoglobulin, purified from pooled milk: size‐exclusion chromatography, CD and NMR measurements were used to study the aggregation and stability of this protein. In spite of the high sequence identity and homology of porcine β‐lactoglobulin with the widely studied bovine species, the two proteins exhibit very different behaviours. The porcine protein shows a monomer–dimer equilibrium with a pH dependence opposite to that observed for the bovine species. Unfolding experiments revealed the presence of an intermediate that probably has excess α helices, as reported for equine species. Modelling studies were performed on bovine, porcine and equine proteins, and, interestingly, electrostatic surface potential calculations led to results consistent with the different dimer interface found for porcine β‐lactoglobulin in the crystal structure. Interaction studies revealed that porcine β‐lactoglobulin is unable to bind fatty acids at any pH, thus questioning the main functional role proposed for lactoglobulins as fatty acid transporters or solubilizers.
Brood Reduction in White Storks Mediated through Asymmetries in Plasma Testosterone Concentrations in Chicks
Authors: Sasvári Lajos , Hegyi Zoltán , Péczely Péter 
Published: March 2003

We hypothesized that increasing chick plasma testosterone concentrations, transmitted from the mothers via their eggs, enhances survival of their offspring and that the fitness of the young, depending on the maternal hormones, is influenced by parental quality. To test our hypotheses we distinguished the broods of white storks Ciconia ciconia L. where chicks died and those where all chicks survived. We analysed the plasma testosterone concentrations in the chicks, the ability of the chicks to be first to receive food and the mass of chicks before fledging in relation to their hatching order and recorded the body mass of parents and food mass delivered by them.Female storks used the asymmetries in testosterone concentrations within a brood to control brood size and adjusted the number of young hatched to match the parental ability to rear offspring. Females of poor condition altered the testosterone concentrations to produce large differences between the chicks: The first‐hatched chicks, which had high plasma testosterone levels, responded faster to the feeding parent and received more food than did their younger siblings. One or two later‐hatched chicks, which had lower testosterone levels, died in these broods. Females in good condition produced small differences in testosterone concentrations between the chicks and all chicks survived in their brood. Chicks that were raised by the females of poor condition in reduced broods were heavier than chicks that were raised by females of good condition in broods where all chicks survived.We suggest that the control of brood size by testosterone concentration, transmitted by the mother to the chicks, is a hormonal means of condition‐dependent reproductive strategy in the white stork.
Aggressiveness in Two Male Morphs of the Bulb Mite Rhizoglyphus robini
Authors: Radwan Jacek , Czyż Magorzata , Konior Magdalena , Kolodziejczyk Maria 
Published: March 2003

We studied differences in aggressiveness between two male morphs of the bulb mite Rhizoglyphus robini (Acari: Acaridae). Heteromorphic males have a thickened and sharply terminating third pair of legs, whereas homeomorphic males have unmodified legs. In another genus (Caloglyphus) of the family Acaridae, modified legs are used during fights to stab (often mortally) other males, but fights have not so far been observed in the genus Rhizoglyphus.In this study we showed that in Rhizoglyphus robini males fight in a way similar to that previously described for the genus Caloglyphus. The presence of females increased the duration of aggressive interactions and caused earlier initiation, but fights were also observed in the absence of females. Pairs of heteromorphic males spent more time on aggressive interactions than did mixed pairs of heteromorphs and homeomorphs or pairs of homeomorphic males. Although the proportion of pairs involved in aggressive interactions did not differ between these three morph combinations, clasping of the opponent with legs, which is a typical behaviour of killer Caloglyphus males that precedes stabbing, occurred more often between pairs of heteromorphs than between mixed pairs and pairs of homeomorphs. This is consistent with our other finding that mortality attributable to male aggression was only recorded in pairs of males containing at least one heteromorphic male. We conclude that the modified legs of heteromorphic R. robini males function as a weapon in aggressive interactions between males and that, as previously described for the genus Caloglyphus, male morphs differ in aggressiveness.
The Role of Mate‐Choice Copying and Disruption Effects in Mate Preference Determination of Limia perugiae (Cyprinodontiformes, Poeciliidae)
Authors: Applebaum Scott L. , Cruz Alexander 
Published: March 2003

AbstractRecent experimental and theoretical evidence suggests that social factors may play an important role in female choice and sexual selection. This is in contrast to the assumption made by most classical sexual selection models that female preferences are entirely determined by genetic factors. This study examined whether female preferences in the poeciliid fish Perugia's Limia (Limia perugiae) are reversible through (i) mate‐choice copying (imitative) behavior, or (ii) a disruption effect (reduced consistency of female preference in response to disruption). Females in binary choice trials were given a mate‐choice copying opportunity by exposing them to a second, model female displaying a preference for a male that was not initially preferred. Females were found to reverse their initial preference significantly more often when presented with an opportunity to observe the model female then in the absence of such an opportunity. In separate trials, females were presented with the opportunity to observe a model female displaying a preference for a male that was initially preferred. No significant difference was observed in the number of reversals between treatments in which females were presented with a copying opportunity that contradicted their initial preference and one that reinforced it. Our results suggest that mate‐choice copying does not contribute significantly to female preferences in L. perugiae and that disruption may reduce the ability of females to choose consistently.
Alarm Calls Affect Foraging Behavior in Eastern Chipmunks (Tamias striatus, Rodentia: Sciuridae)
Authors: Baack Jessica K. , Switzer Paul V. 
Published: March 2003

AbstractWe used playback experiments to test whether alarm calls affected the foraging behavior of eastern chipmunks (Tamias striatus). We subjected chipmunks, foraging at artificial feeding stations, to three playback treatments (silent, control noise, and alarm call) and examined changes in vigilant and foraging behavior. Chipmunks responded to alarm calls with a greater degree and duration of vigilant behavior, such as look‐ups and alert postures. Chipmunks also ran a shorter distance to cover and took longer to re‐emerge from the burrow after hearing an alarm call. Alarm calls caused individuals to spend more time exposed at the feeding stations; however, individuals also took significantly fewer seeds after hearing an alarm call. This was not due to a difference in the time spent handling food, but rather to a slower rate of loading. Chipmunks appear to sacrifice energy gain by increasing vigilance after hearing an alarm call. This study suggests that to avoid the costs of unnecessary escape behavior, individuals directly assess their own risk rather than relying only on indirect cues such as alarm calls.
Size‐Dependent Chemosensory Responses to Familiar and Unfamiliar Conspecific Faecal Pellets by the Iberian Rock‐Lizard, Lacerta monticola
Authors: Aragón Pedro , López Pilar , Martín José 
Published: March 2003

AbstractWe conducted a field study to analyse the social relationships between males of the Iberian rock lizard (Lacerta monticola). The degree of familiarity was determined based on the degree of overlap between their home ranges. We then designed a laboratory experiment to test whether the same males were able to discriminate between familiar and unfamiliar conspecifics using faecal pellet odours. Differential tongue‐flick rates suggest that large males (snout‐to‐vent length, SVL > 75 mm), at least, may discriminate between odours of familiar and unfamiliar males. The behavioural responses were dependent on relative differences in body size between the responding male and the male that donated the faecal pellet. Thus, as responding small males increased in size relative to their corresponding familiar male, their rate of tongue‐flicking significantly decreased; this was not the case in response to unfamiliar males. In contrast, there were no significant correlations between the response of large males to familiar or unfamiliar male stimuli, regardless of size differences. These results suggest that chemical cues contained in faecal pellets allow individual recognition in male L. monticola, and that the response depends on body size. We suggest that faecal pellets might be used to scent‐mark home ranges, which would contribute to lowering the costs of aggressive interactions.
Tail Length and Mutual Mate Choice in Bearded Tits (Panurus biarmicus)
Authors: Romero‐Pujante Mariló , Hoi Herbert , Blomqvist Donald , Valera Francisco 
Published: March 2003

AbstractDirect sexual selection via mutual mate choice can result in both sexes showing conspicuous traits. We experimentally tested whether this hypothesis can explain tail length in the bearded tit (Panurus biarmicus). In this species, both sexes have a long, graduated tail. Males have, however, a longer tail than females, suggesting perhaps that females are choosier than males in selecting mates. We used two choice set‐ups for each sex: shortened vs. control tail individuals and elongated vs. control tail individuals. We found that direct sexual selection seems to operate differently in the two sexes. In both set‐ups, females spent more time with the male with the longest tail, and they also showed sexual display behaviour only towards these males. Males spent more time with control than with short‐tailed females, but they did not discriminate between control and long‐tailed females. Moreover, males displayed preference towards both short‐ and long‐tailed females. Thus, females preferred long‐tailed males, whereas males did not always prefer long‐tailed females. Our study suggests that mutual mate choice has played a role in the evolution of long tails in bearded tits. It also suggests that the sexual dimorphism in tail length has evolved because mate choice exerts a stronger sexual selection pressure on males than on females.
Distribution and ecology of Armillaria species in Greece
Authors: Tsopelas P. 
Published: March 2003

SummaryFive Armillaria species were identified in a nationwide survey in Greece. Armillaria mellea was present in coniferous and broad‐leaved forests in most of the areas examined, except the high altitudes (above 1100 m) of the mountains of north Greece. It was found to cause significant damage in fir forests as well as in fruit orchards and vineyards. Armillaria gallica was common in coniferous and broad‐leaved forests in the high altitudes of central and northern Greece, predominating in the beech forests. The fungus was a weak parasite or a saprophyte of forest trees and was occasionally found on cultivated plants. Armillaria ostoyae was not found in southern and central parts of the country, but it has a wide distribution in the mountain forests of northern Greece and causes significant damage on fir, black pine, Scots pine and spruce. Armillaria cepistipes was recorded at high altitudes (1400–1800 m) on two mountains of northern Greece, mostly as a saprophyte in coniferous and broad‐leaved forests. Armillaria tabescens was rare in the forests of Greece; it was found to cause disease in almond tree orchards.