bluuebunny/crossref_metadata_2025 huggingface dataset:
Economics and the design of nature conservation policy: a case study of wild goose conservation in Scotland using choice experiments
Authors: Hanley Nick , MacMillan Douglas , Patterson Ian , Wright Robert E.
Published: May 2003

This paper applies the ‘choice experiment’ method to investigate public preferences over the design of wild goose conservation policy in Scotland. We argue that this method can shed useful light on the design of conservation policy, allowing policy‐makers to take account of people's preferences, be they members of the general public (whose taxes often pay for conservation actions), local residents more directly affected by the policy, or visitors to wildlife areas. Preferences can be quantified in economic terms, so that the costs and benefits of different policy designs can be compared. In our study, we find that the general public, local residents and visitors have very different preferences for the conservation of geese. Whether geese are shot, the endangered status of geese, the spatial targeting of conservation and the size of the goose population all have impacts on the perceived benefits of conservation. In general, though, people are willing to pay for wild geese conservation.
Crouching tigers, hidden prey: Sumatran tiger and prey populations in a tropical forest landscape
Authors: O'Brien Timothy G. , Kinnaird Margaret F. , Wibisono Hariyo T.
Published: May 2003

We examine the abundance and distribution of Sumatran tigers (Panthera tigris sumatrae) and nine prey species in Bukit Barisan Selatan National Park on Sumatra, Indonesia. Our study is the first to demonstrate that the relative abundance of tigers and their prey, as measured by camera traps, is directly related to independently derived estimates of densities for these species. The tiger population in the park is estimated at 40–43 individuals. Results indicate that illegal hunting of prey and tigers, measured as a function of human density within 10 km of the park, is primarily responsible for observed patterns of abundance, and that habitat loss is an increasingly serious problem. Abundance of tigers, two mouse deer (Tragulus spp.), pigs (Sus scrofa) and Sambar deer (Cervus unicolor) was more than four times higher in areas with low human population density, while densities of red muntjac (Muntiacus muntjac) and pigtail macaques (Macaca nemestrina) were twice as high. Malay tapir (Tapirus indicus) and argus pheasant (Argusianus argus), species infrequently hunted, had higher indices of relative abundance in areas with high human density. Edge effects associated with park boundaries were not a significant factor in abundance of tigers or prey once human density was considered. Tigers in Bukit Barisan Selatan National Park, and probably other protected areas throughout Sumatra, are in imminent danger of extinction unless trends in hunting and deforestation are reversed.
Science deficiency in conservation practice: the monitoring of tiger populations in India
Authors: Karanth K. Ullas , Nichols James D. , Seidenstricker John , Dinerstein Eric , Smith James L. David , McDougal Charles , Johnsingh A. J. T. , Chundawat Raghunandan S. , Thapar Valmik
Published: May 2003

Conservation practices are supposed to get refined by advancing scientific knowledge. We study this phenomenon in the context of monitoring tiger populations in India, by evaluating the ‘pugmark census method’ employed by wildlife managers for three decades. We use an analytical framework of modern animal population sampling to test the efficacy of the pugmark censuses using scientific data on tigers and our field observations. We identify three critical goals for monitoring tiger populations, in order of increasing sophistication: (1) distribution mapping, (2) tracking relative abundance, (3) estimation of absolute abundance. We demonstrate that the present census‐based paradigm does not work because it ignores the first two simpler goals, and targets, but fails to achieve, the most difficult third goal. We point out the utility and ready availability of alternative monitoring paradigms that deal with the central problems of spatial sampling and observability. We propose an alternative sampling‐based approach that can be tailored to meet practical needs of tiger monitoring at different levels of refinement.
Effects of introduced Rattus rattus on endemic small mammals in dry deciduous forest fragments of western Madagascar
Authors: Ganzhorn Jörg U.
Published: May 2003

Studies on the effects of nonindigenous invasive species and habitat fragmentation on native ecosystems have become priorities for conservation biologists. This is relevant particularly for islands with high degrees of endemism. In the present paper the role of fragmentation and possible effects of introduced Rattus rattus on endemic rodents (Macrotarsomys bastardi and Eliurus spp.) and the gray mouse lemur (Microcebus murinus) were assessed for dry deciduous forests in Madagascar. Capture rates of endemic rodents declined significantly with declining fragment size. Macrotarsomys bastardi was never caught in primary forest fragments < 600 ha or outside the primary forest but captures of M. bastardi were correlated to changes in vegetation structure associated with fragmentation rather than to fragment size per se. Eliurus spp. were also found in small primary‐forest fragments and in secondary forests close to primary forests, suggesting that secondary forests can serve as buffer zones or corridors for this species. The lemur M. murinus was caught in all types of primary and secondary forest. This species did not seem to respond to fragmentation of primary forests. Even though it was found in most secondary‐forests, capture rates declined as these secondary forest formations increased in size. This indicates that secondary forests are suboptimal habitats for M. murinus. Rattus rattus were caught in all vegetation formations except for the open savanna. Their capture rates increased as forest fragments became smaller, indicating that mosaics of different habitat types are beneficial for introduced rats. There was no significant relation between capture rates of R. rattus and the endemic species. In conclusion, these results suggest that (1) native Malagasy rodents of the dry deciduous forest are more sensitive towards forest disturbance than M. murinus; (2) there is no indication for negative interactions between introduced rats and the native small‐mammal fauna.
Managing puma hunting in the western United States: through a metapopulation approach
Authors: Laundré John , Clark Tim W.
Published: May 2003

To achieve long‐term viability of hunted puma (Puma concolor) populations (even at historically low densities), we propose a management plan based on the metapopulation concept that designatessource areas(closed to hunting) andsink areas(open to hunting). We use 11 years of data from Idaho and Utah to demonstrate how the proposed management plan might be implemented. We use minimum and maximum densities of resident animals to calculate minimum and maximum effective population sizes, neighbourhood areas (regional management units) and usable habitat within the units. We designate sink and source areas based on their size, accessibility to hunters and juxtaposition. We show that closing 63% of puma habitat to hunting would ensure long‐term puma population viability while permitting traditional hunting levels in other areas. This system could be adapted to existing state (and interstate) hunting management units, and we outline several steps by which wildlife agencies might set up a process (including public participation) to manage puma hunting.
Umbrella species: critique and lessons from East Africa
Authors: Caro T. M.
Published: May 2003

Umbrella species are ‘species with large area requirements, which if given sufficient protected habitat area, will bring many other species under protection’. Historically, umbrella species were employed to delineate specific reserve boundaries but are now used in two senses: (1) as aids to identifying areas of species richness at a large geographic scale; (2) as a means of encompassing populations of co‐occuring species at a local scale. In the second sense, there is a dilemma as to whether to maximize the number or viability of background populations; the umbrella population itself needs to be viable as well. Determining population viability is sufficiently onerous that it could damage the use of umbrella species as a conservation shortcut. The effectiveness of using the umbrella‐species concept at a local scale was investigated in the real world by examining reserves in East Africa that were gazetted some 50 years ago using large mammals as umbrella species. Populations of these species are still numerous in most protected areas although a few have declined. Populations of other, background species have in general been well protected inside reserves; for those populations that have declined, the causes are unlikely to have been averted if reserves had been set up using other conservation tools. Outside one reserve, Katavi National Park in Tanzania, background populations of edible ungulates and small carnivores are lower than inside the reserve but small rodent and insectivore abundance is higher. While we cannot compare East African reserves to others not gazetted using umbrella species, the historical record in this region suggests that umbrella species have been an effective conservation shortcut perhaps because most reserves were initially large and could encompass substantial populations of background species. It is therefore premature to discard the local‐scale umbrella‐species concept despite its conceptual difficulties.
Indonesia's threatened birds: over 500 million years of evolutionary heritage at risk
Authors: Mooers A. Ø , Atkins R. A.
Published: May 2003

Using published IUCN red‐lists for the threatened bird species of Indonesia (Stattersfield & Capper, 2000), we document that the species at risk in Indonesia are not a taxonomically random sample, consistent with patterns seen at the global scale (Purvis et al., 2000). This implies that a greater than random proportion of Indonesia's and the world's (through the loss of endemics) phylogenetic diversity (PD) is at risk (Von Euler, 2001). Using cytochrome b (Johns & Avise, 1998) and a distance‐based taxonomy, we attempt to quantify this projected loss of PD in millions of years and conclude with a brief discussion of the conservation implications for Indonesia, and of the use of ‘evolutionary heritage’ as a measure for conservation at the geopolitical level.
The early tissue response to titanium and LactoSorb screws
Authors: Mollaoglu Nur , Çetiner Sedat , Alpaslan Cansu , Gültekin Sibel Elif , Alpar Reha
Published: May 2003

Abstract –  Fractures of the maxillofacial region are common in the elderly people. Titanium and LactoSorb screws are the widely accepted materials for use in the maxillofacial fractures. This study was undertaken to evaluate the early tissue response following the insertion of both titanium and LactoSorb screws composed of 82% PLLA and 18% PGA in an elderly animal model. In this study, 22 titanium and 22 LactoSorb screws were applied to calvaria of 44 guinea pigs that were 10–11 months old. Animals were sacrificed on postoperative days 3, 7, 14, 30 and 60. Screws were retrieved with surrounding bone tissue and the specimens were prepared for routine histologic examination. All the specimens were evaluated by light microscopy. Histometric analysis revealed that there was no significant difference between LactoSorb and titanium screws for the new bone formation. The biodegradation of LactoSorb screws was not complete by the end of day 60. In conclusion, both materials were well tolerated and induced bone formation without causing adverse tissue response in an elderly animal model. Our results suggest that both LactoSorb and titanium miniplates and screws can be used safely, regardless of the increasing age. However, LactoSorb may be the first choice as it does not require a second operation for removal and has late biodegradation in elderly that keeps its support for a relatively longer time during fracture healing.
Treatment of a non‐vital immature incisor with mineral trioxide aggregate (MTA)
Authors: Maroto Myriam , Barbería Elena , Planells Paloma , Vera Vicente
Published: May 2003

Abstract  – A case of severe crown fracture and luxation in the upper permanent incisors of a 9‐year‐old boy is reported. The treatment of one of the injured teeth included apexification with calcium hydroxide and endodontic treatment with gutta‐percha obturation. The other incisor was also treated with calcium hydroxide, but as there was no apical stop after 3 years of treatment, it was decided to use a new root‐end filling material: mineral trioxide aggregate (MTA). At follow‐up 12 months later, the tooth was asymptomatic and radiographically showed the initial repair of the radiolucent apical lesion.
Periodontal mechano‐sensory responses following trauma to permanent incisor teeth in children
Authors: Gaubert S. A. , Hector M. P.
Published: May 2003

Abstract –  The aim of this study was to investigate mechano‐sensory responses of injured and uninjured incisor teeth. Twenty‐five children who had experienced dental trauma, together with age‐ and gender‐matched controls, were studied prospectively. Touch thresholds of incisor teeth from both groups were determined using calibrated sets of von Frey hairs (force range 0.5–10.0 g in 0.5 g increments) using a forced choice staircase method. Forces were applied perpendicular to the buccal enamel surfaces along the midline, 2 mm from the incisal edge. Touch threshold was defined as the lowest force detected in three out of the five occasions. Following statistical analysis, P < 0.05 was considered significant. At initial examination, the touch threshold values of 25 traumatised teeth were significantly greater than the untraumatised controls (P < 0.001), and these values approached those of the control teeth over 3–12 months (P > 0.05). Dental trauma was associated with increased touch thresholds in permanent incisor teeth, with recovery toward healthy control values usually occurring between 3–12 months.
Prevalence of traumatic crown fractures to permanent incisors in a childhood population: Móstoles, Spain
Authors: Tapias Miguel Angel , Jiménez‐García Rodrigo , Lamas Fernando , Gil Angel A.
Published: May 2003

Abstract  – The aim of this study was to ascertain the prevalence of traumatic crown fractures in a childhood population and identify the risk factors associated with the same. A cross‐sectional study was conducted, covering 470 schoolchildren and surveying socio‐demographic variables, overweight, overbite, number of teeth, cause, setting, season, type of injury and restoration. We measured prevalence, performed univariate and multivariate analyses of risk factors for crown fracture, and calculated the Odds Ratio (OR) and its 95% confidence intervals (95% CI). Prevalence of traumatic crown fractures to permanent incisors was 17.4% (95% CI: 14–20.8). Falls were the most frequent cause of dental trauma (43.9%). Boys and children with overbite registered a 2.13‐ and 1.81‐fold higher risk of crown fractures, respectively. Owing to high prevalence, crown fractures in schoolchildren aged 10 years constitute a public health problem. Being male and having overbite are risk factors for crown fracture. Healthcare promotion and education at health centres is called for to prevent such injuries.
External and occlusal trauma to dental implants and a case report
Authors: Flanagan Dennis
Published: May 2003

Abstract –  Dental implants subjected to traumatic forces can survive. Cortical bone seems to provide a protective energy‐absorbing mechanism in the collagen polymer that helps to prevent microcracking and fracture of bone. The collagen polymer has cross‐linking bonds that break and absorb the energy of a traumatic impact so as not to cause damage to the main polymer chain. A case reported demonstrates that a traumatic force damaged the implant prosthetic crown, but not the bone encasing the implant or the integration of the implant.
External inflammatory and replacement resorption of luxated, and avulsed replanted permanent incisors: a review and case presentation
Authors: Finucane David , Kinirons Martin J.
Published: May 2003

Abstract –  External inflammatory resorption and replacement resorption are complicating factors that may result from traumatic dental injuries when the tooth is luxated or avulsed and replanted. Resorption may, ultimately, result in loss of the tooth. However, with appropriate treatment, the prognosis for these teeth is greatly improved, with the possibility of preventing or arresting resorption. The purpose of this paper is to review these trauma entities, to discuss factors that influence the occurrence of resorption and to describe the most appropriate treatment. A case is presented, illustrating both resorption entities, but with varying outcomes.
Root fracture detection on digital images: effect of the zoom function
Authors: Kositbowornchai Suwadee , Sikram Supaporn , Nuansakul Rattanon , Thinkhamrop Bandit
Published: May 2003

Abstract ‐ Objectives: To assess the accuracy of root fracture detection at various levels of image magnification.

Methods: Extracted single‐rooted human teeth (100 non‐fractured and 101 fractured) were mounted singly in wax blocks and a charge‐coupled device (CCD)‐based digital Sidexis imaging system was used to take images using the paralleling technique. Image sizes 1 : 1, 2 : 1, and 1 : 2 were prepared for each tooth. All 603 images were examined randomly and interpreted by an experienced radiologist unaware of which images were of fractured roots. The degree of agreement between image interpretation and the actual condition was estimated using a kappa statistic. A modified goodness‐of‐fit χ2‐test was used to assess the significance of the difference between the three image sizes. The sensitivity, specificity, predictive value, and likelihood ratio were used to evaluate the accuracy of root fracture detection at each level of magnification. The degree of agreement between pairs of viewing conditions was also calculated using a kappa statistic.

Results: There was substantial agreement between image interpretation and the actual condition for both the 1 : 1 and 1 : 2 image sizes but only moderate agreement for the 2 : 1 size. The kappa values (and 95% CIs) were 0.65 (0.51–0.79), 0.63 (0.49–0.77), and 0.58 (0.44–0.72), respectively. The differences were not statistically significant (P‐value = 0.79). The sensitivity (95% CI) of the image magnification ranged between 79.0% (70.0–87.5%) and 81.0% (72.2–87.5%). The 1 : 1 image size gave the highest specificity of 86.1% (95% CI: 78.1–91.6%). The positive predictive values (95% CI) of the 1 : 1, 1 : 2, and 2 : 1 images sizes were 84.9% (76.3–90.8%), 81.8% (73.1–88.2%), and 77.9% (69.0–84.8%), respectively. The negative predictive values (95% CI) were 80.6% (72.1–86.9%), 81.4% (73.8–88.9%), and 80.4% (71.4–87.1%) for the 1 : 1, 1 : 2, and 2 : 1 image sizes, respectively. The likelihood ratios of the positive test (95% CI) were 5.70 (3.47–9.36), 4.55 (3.00–6.98), and 3.56 (2.45–5,16) for the 1 : 1, 1 : 2, and 2 : 1 image sizes, respectively. The likelihood ratios of the negative test (95% CI) were 0.24 (0.17–0.36), 0.23 (0.15–0.35), and 0.25 (0.16–0.37) for the 1 : 1, 1 : 2, and 2 : 1 image sizes, respectively. The reliability between pairs of viewing conditions was substantial. The kappa statistics (95% CI) was 0.80 (0.72–0.88), 0.73 (0.64–0.83), and 0.77 (0.68–0.86), respectively.

Conclusion: In this in vitro study, the three digital magnifications used did not affect the detection of root fractures.
Single‐tooth implant treatment in the anterior region of the maxilla for treatment of tooth loss after trauma: a retrospective clinical and interview study
Authors: Andersson Lars , Emami‐Kristiansen Zina , Högström Jan
Published: May 2003

Abstract –  The aim of this study was to evaluate the results of single‐tooth implant treatment in patients where teeth have been lost as a result of trauma. Also, the patients' and professionals' opinions regarding the final outcome of treatment were assessed. Thirty‐four patients with 42 lost teeth were evaluated by clinical and radiographic examinations and interviews 2–5 years after treatment. A professional who had not taken part in the treatment evaluated the implant crowns. Central maxillary incisors were the most frequently lost and replaced teeth after trauma (75%) followed by lateral incisors (21%). In patients with incomplete growth, implant treatment was generally postponed until completion of growth. Lack of space was treated by presurgical orthodontics (7%) or by selecting an implant with a reduced diameter (5%). Deficiency of bone was seen in 17% and was treated by bone grafting or local augmentation prior to implant surgery. Patients who had lost two or more teeth after trauma were all subjected to bone grafting. Preservation of roots in the alveolar process seemed to maintain the bone volume enabling better conditions for later implant placement. Forty‐one implants (97.6%) were integrated successfully. Complications were few and of minor importance (9.5% before and 12% after cementation of crowns) and could all be managed. No or minimal bone loss was seen. In general, the patients felt that they received good care and that they were well informed about their treatment. Some patients reported that the local anesthesia procedure was not pain‐free, but 71% of the patients experienced the treatment as pain‐free. For each of the variables (color, shape, height, and size of the crowns), the highest degree of satisfaction was noted in 93–98% of the patients and 91–95% of the single evaluating professional. Given that the patients have finished growth and a careful treatment planning and timing are performed, the functional and esthetical outcome of single‐tooth implant treatment today is excellent and can be recommended for replacing tooth losses after trauma in the anterior region of the maxilla.
Bidirectional integrin alphaIIbbeta3 signalling regulating platelet adhesion under flow: contribution of protein kinase C
Authors: GIULIANO Simon , NESBITT Warwick S. , ROONEY Michael , JACKSON Shaun P.
Published: May 2003

Platelet adhesion on von Willebrand factor (vWf) requires the co-ordinated adhesive function of glycoprotein Ib/V/IX and integrin αIIbβ3. Recent evidence [Nesbitt, Kulkarni, Giuliano, Gonclaves, Dopheide, Yap, Harper, Salem and Jackson (2002) J. Biol. Chem. 277, 2965–2972] suggests that outside-in signals from both receptors play important roles in regulating platelet-adhesion dynamics under flow. In the present study, we have examined the mechanisms utilized by protein kinase C (PKC) to promote irreversible platelet adhesion on vWf. We demonstrate that PKC is primarily activated downstream of integrin αIIbβ3, not glycoprotein Ib, during platelet adhesion on vWf. This integrin αIIbβ3-dependent PKC activation establishes a positive-feedback loop that promotes further integrin αIIbβ3 activation, calcium mobilization and firm platelet adhesion. This feedback loop appears to be most relevant at relatively low cytosolic calcium concentrations (mean Δ[Ca2+]i~100 nM) as artificially elevating calcium (mean Δ[Ca2+]i > 500 nM) induces integrin αIIbβ3 activation and irreversible platelet adhesion independent of PKC. Our studies demonstrate the existence of a complex signalling relationship operating between PKC, cytosolic calcium and integrin αIIbβ3 that serves to regulate platelet-adhesion dynamics under flow. Furthermore, we have established the existence of PKC-dependent and -independent pathways regulating integrin αIIbβ3 activation and stable platelet adhesion on vWf.
U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor of 65 kDa, U2AF65, can promote U1 snRNP recruitment to 5′ splice sites
Authors: FÖRCH Patrik , MERENDINO Livia , MARTÍNEZ Concepción , VALCÁRCEL Juan
Published: May 2003

The splicing factor U2AF65, U2 small nuclear ribonucleoprotein particle (snRNP) auxillary factor of 65 kDa, binds to pyrimidine-rich sequences at 3′ splice sites to recruit U2 snRNP to pre-mRNAs. We report that U2AF65 can also promote the recruitment of U1 snRNP to weak 5′ splice sites that are followed by uridine-rich sequences. The arginine- and serine-rich domain of U2AF65 is critical for U1 recruitment, and we discuss the role of its RNA–RNA annealing activity in this novel function of U2AF65.
Engineered allosteric mutants of the integrin alphaMbeta2 I domain: structural and functional studies
Authors: McCLEVERTY Clare J. , LIDDINGTON Robert C.
Published: May 2003

The α-I domain, found in the α-subunit of the leucocyte integrins such as αMβ2 and αLβ2, switches between the open and closed tertiary conformations, reflecting the high- and low-affinity ligand-binding states of the integrin that are required for regulated cell adhesion and migration. In the present study we show, by using point mutations and engineered disulphide bonds, that ligand affinity can be reduced or increased allosterically by altering the equilibrium between the closed and open states. We determined equilibrium constants for the binding of two ligands, fibrinogen and intercellular cell-adhesion molecule 1, to the αM-I domain by surface plasmon resonance, and determined crystal structures of a low-affinity mutant. Locking the domain in the open conformation increases affinity by a factor of no greater than 10, consistent with a closely balanced equilibrium between the two conformations in the absence of ligand. This behaviour contrasts with that of the unliganded αL-I domain, for which the equilibrium lies strongly in favour of the closed conformation. These results suggest significant differences in the way the parent integrins regulate I domain conformation and hence ligand affinity.
SEA (sea-urchin sperm protein, enterokinase and agrin)-module cleavage, association of fragments and membrane targeting of rat intestinal mucin Muc3
Authors: KHATRI Ismat A. , WANG Rongquan , FORSTNER Janet F.
Published: May 2003

In a previous study we showed, by transient expression studies in COS-1 cells, that the C-terminal domain of rat intestinal membrane mucin Muc3 was cleaved between glycine and serine within a GSIVV (one-letter) amino acid sequence during its residence in the endoplasmic reticulum. The extracellular domain fragment remained linked to the membrane-associated fragment by non-covalent interactions. The present study demonstrates that cleavage depends not only on the presence of the G/SIVV site (where G/S is the glycine↓serine cleavage site), but also on more distant C-terminal sequences in the SEA (sea-urchin sperm protein, enterokinase and agrin) module. Inhibition of N-glycosylation by tunicamycin treatment of transfected cells did not prevent re-association of fragments, although cleavage was partially impaired, as some of the non-glycosylated, non-cleaved products were seen to accumulate in cells. Membrane targeting of the Muc3 domain and its cleavage products occurred in transfected cells and was not impaired in mutants in which the cleavage site was mutated. Targeting was also not impaired for products devoid of N-linked oligosaccharides. Our studies thus indicate that (a) cleavage within the SEA module of rat Muc3 requires participation of peptide sequences located C-terminal of and distant from the cleavage site, (b) re-association of the fragments requires the SEA module, but is independent of N-linked oligosaccharides, and (c) membrane targeting of the mucin is independent of the SEA-module-cleavage reaction.
Partially esterified oligogalacturonides are the preferred substrates for pectin methylesterase of Aspergillus niger
Authors: van ALEBEEK Gert-Jan W. M. , van SCHERPENZEEL Katrien , BELDMAN Gerrit , SCHOLS Henk A. , VORAGEN Alphons G. J.
Published: May 2003

Investigations on the mode of action of Aspergillus niger pectin methylesterase (PME) towards differently C6- and C1-substituted oligogalacturonides (oligoGalpA) are described. De-esterification of methyl-esterified (un)saturated oligoGalpA proceeds via a specific pattern, depending on the degree of polymerization. Initially, a first methyl ester of the oligomer is hydrolysed, resulting in one free carboxyl group. Subsequently, this first product is preferred as a substrate and is de-esterified for a second time. This product is then accumulated and hereafter de-esterified further to the final product, i.e. oligoGalpA containing one methyl ester located at the non-reducing end residue for both saturated and unsaturated oligoGalpA, as found by post-source decay matrix-assisted laser-desorption/ionization–time-of-flight MS. The saturated hexamer is an exception to this: three methyl esters are removed very rapidly, instead of two methyl esters. When unsaturated oligoGalpA were used, the formation of the end product differed slightly, suggesting that the unsaturated bond at the non-reducing end influences the de-esterification process. In vivo, PME prefers methyl esters, but the enzyme appeared to be tolerant for other C6- and C1-substituents. Changing the type of ester (ethyl esterification) or addition of a methyl glycoside (C1) only reduced the activity or had no effect respectively. The specific product pattern was identical for all methyl- and ethyl-esterified oligoGalpA and methyl-glycosidated oligoGalpA, which strongly indicates that one or perhaps two non-esterified oligoGalpA are preferred in the active-site cleft.
High-mobility group A1 proteins are overexpressed in human leukaemias
Authors: PIERANTONI Giovanna Maria , AGOSTI Valter , FEDELE Monica , BOND Heather , CALIENDO Irene , CHIAPPETTA Gennaro , COCO Francesco LO , PANE Fabrizio , TURCO Maria Caterina , MORRONE Giovanni , VENUTA Salvatore , FUSCO Alfredo
Published: May 2003

High-mobility group A (HMGA) proteins are non-histone nuclear proteins that bind DNA and several transcription factors. They are involved in the regulation of chromatin structure and function. HMGA protein expression is low in normal adult tissues, but abundant during embryonic development and in several human tumours. Rearrangements of the HMGA genes have been frequently detected in human benign tumours of mesenchymal origin, e.g. lipomas, lung hamartomas and uterine leiomiomas. HMGA proteins have been implicated in the control of cell growth and differentiation of the pre-adipocytic cell line 3T3-L1. In an attempt to better understand the role of HMGA1 proteins in haematological neoplasias and in the differentiation of haematopietic cells, we have investigated their expression in human leukaemias and in leukaemic cell lines induced to terminal differentiation. Here we report HMGA1 overexpression in most fresh human leukaemias of different origin and in several leukaemic cell lines. Moreover, differentiation of three cell lines towards the megakaryocytic phenotype was associated with HMGA1 protein induction, whereas induction of erythroid and monocytic differentiation generally resulted in reduced HMGA1 expression.
Characterization of poultry egg-white avidins and their potential as a tool in pretargeting cancer treatment
Authors: HYTÖNEN Vesa P. , LAITINEN Olli H. , GRAPPUTO Alessandro , KETTUNEN Anu , SAVOLAINEN Janne , KALKKINEN Nisse , MARTTILA Ari T. , NORDLUND Henri R. , NYHOLM Thomas K. M. , PAGANELLI Giovanni , KULOMAA Markku S.
Published: May 2003

Chicken avidin and bacterial streptavidin are proteins used in a wide variety of applications in the life sciences due to their strong affinity for biotin. A new and promising use for them is in medical pretargeting cancer treatments. However, their pharmacokinetics and immunological properties are not always optimal, thereby limiting their use in these applications. To search for potentially beneficial new candidates, we screened egg white from four different poultry species for avidin. Avidin proteins, isolated from the duck, goose, ostrich and turkey, showed a similar tetrameric structure, similar glycosylation and stability against both temperature and proteolytic activity of proteinase K as chicken avidin. Biotin-binding properties of these avidins, measured using IAsys optical biosensor, were similar to those found in avidin from the chicken. Three of these novel avidins, however, showed different immunological cross-reactivities when compared with chicken avidin. The patient sera responses to duck, goose and ostrich avidins were also lower than those observed for chicken and turkey avidins. Our findings suggest that the use of these proteins offers advantages over chicken avidin and bacterial streptavidin in pretargeting applications.
Prion peptide induces neuronal cell death through a pathway involving glycogen synthase kinase 3
Authors: PÉREZ Mar , ROJO Ana I. , WANDOSELL Francisco , DÍAZ-NIDO Javier , AVILA Jesús
Published: May 2003

Prion diseases are characterized by neuronal cell death, glial proliferation and deposition of prion peptide aggregates. An abnormal misfolded isoform of the prion protein (PrP) is considered to be responsible for this neurodegeneration. The PrP 106–126, a synthetic peptide obtained from the amyloidogenic region of the PrP, constitutes a model system to study prion-induced neurodegeneration as it retains the ability to trigger cell death in neuronal cultures. In the present study, we show that the addition of this prion peptide to cultured neurons increases the activity of glycogen synthase kinase 3 (GSK-3), which is accompanied by the enhanced phosphorylation of some microtubule-associated proteins including tau and microtubule-associated protein 2. Prion peptide-treated neurons become progressively atrophic, and die ultimately. Both lithium and insulin, which inhibit GSK-3 activity, significantly decrease prion peptide-induced cell death both in primary neuronal cultures and in neuroblastoma cells. Finally, the overexpression of a dominant-negative mutant of GSK-3 in transfected neuroblastoma cells efficiently prevents prion peptide-induced cell death. These results are consistent with the view that the activation of GSK-3 is a crucial mediator of prion peptide-induced neurodegeneration.
Calcium and domain interactions contribute to the thermostability of domains of the multimodular cellobiohydrolase, CbhA, a subunit of the Clostridium thermocellum cellulosome
Authors: KATAEVA Irina A. , UVERSKY Vladimir N. , LJUNGDAHL Lars G.
Published: May 2003

Each of three internal domains of multi-modular cellobiohydrolase CbhA from Clostridium thermocellum, X11, X12 (previously designated as fibronectin type 3-like modules, Fn31 and Fn32) and family 3 carbohydrate-binding module (CBM3) binds 1 mol of Ca2+. Structures and thermal stabilities of X11, X12, CBM3, X11X12, and X11X12-CBM3 containing Ca2+ (holo-proteins) and without Ca2+ (apo-proteins) have been studied using CD spectroscopy. All domains are β-proteins with irregular far-UV CD spectra due to the aromatic side chain contributions. The positive signal at 294 nm in the near-UV CD spectrum of X11 lacking a tryptophan residue might be attributed to the presence of aromatic clusters. Thermal denaturation of all proteins is reversible and results in the total loss of tertiary structure and preservation of significant amount of ordered secondary structure. Removal of Ca2+ destabilizes polypeptides in a different way and to a different extent. It decreases the melting temperature (Tm) (by 20 °C) and co-operativity of thermal transition of X11, increases the number of transitions and lowers the co-operativity of unfolding of CBM3, and slightly decreases Tms (2.4–4.2 °C) of X12, X11X12, and X11X12-CBM3. Transitions of X11X12 and X11X12-CBM3 follow a two-state model regardless of the presence of Ca2+. X11 is strongly stabilized in the apo-X11X12 and apo-X11X12-CBM3 as they display Tms similar to those of individual and combined holo-modules. Observed CD spectra of X11X12 and X11X12-CBM3 differ from those calculated as the simple weighted sum of individual modules. These differences are more prominent in spectra of apo-proteins. The results indicate the presence of inter-domain interactions in CbhA. Holo-modules, i.e. containing Ca2+, behave essentially independently, but in the absence of Ca2+ domain interactions are more important for the conformation of the polypeptides.
Signalling specificity of Ser/Thr protein kinases through docking-site-mediated interactions
Authors: BIONDI Ricardo M. , NEBREDA Angel R.
Published: May 2003

Signal transduction pathways use protein kinases for the modification of protein function by phosphorylation. A major question in the field is how protein kinases achieve the specificity required to regulate multiple cellular functions. Here we review recent studies that illuminate the mechanisms used by three families of Ser/Thr protein kinases to achieve substrate specificity. These kinases rely on direct docking interactions with substrates, using sites distinct from the phospho-acceptor sequences. Docking interactions also contribute to the specificity and regulation of protein kinase activities. Mitogen-activated protein kinase (MAPK) family members can associate with and phosphorylate specific substrates by virtue of minor variations in their docking sequences. Interestingly, the same MAPK docking pocket that binds substrates also binds docking sequences of positive and negative MAPK regulators. In the case of glycogen synthase kinase 3 (GSK3), the presence of a phosphate-binding site allows docking of previously phosphorylated (primed) substrates; this docking site is also required for the mechanism of GSK3 inhibition by phosphorylation. In contrast, non-primed substrates interact with a different region of GSK3. Phosphoinositide-dependent protein kinase-1 (PDK1) contains a hydrophobic pocket that interacts with a hydrophobic motif present in all known substrates, enabling their efficient phosphorylation. Binding of the substrate hydrophobic motifs to the pocket in the kinase domain activates PDK1 and other members of the AGC family of protein kinases. Finally, the analysis of protein kinase structures indicates that the sites used for docking substrates can also bind N- and C-terminal extensions to the kinase catalytic core and participate in the regulation of its activity.
Characterization of SUMO-conjugating enzyme mutants in Schizosaccharomyces pombe identifies a dominant-negative allele that severely reduces SUMO conjugation
Authors: HO Jenny C. Y. , WATTS Felicity Z.
Published: May 2003

The phenotypes of mutants defective in the Schizosaccharomyces pombe SUMO (small, ubiquitin-like modifier)-conjugating enzyme Hus5 (the homologue of Ubc9) show that it is required for recovery from S-phase arrest. Unlike the case with ubiquitination, where ligases are required, SUMO-conjugating enzymes are sufficient for substrate recognition and conjugation of SUMO on to target proteins, at least in vitro. Thus SUMO-conjugating enzymes are likely to be important regulators of sumoylation. Here, we report on the characterization of two hus5 alleles. Although hus5.17 and hus5.62 respond in a similar manner to UV and ionizing radiation, they have different responses to the DNA-synthesis inhibitor, hydroxyurea. In addition, SUMO (Pmt3) is mislocalized in hus5.17 cells, but not in hus5.62 mutant cells. The mutations in hus5.62 and hus5.17 map to Ala129 and the 5′ splice site of intron 2 respectively. We have characterized the Hus5.62 protein and shown, in vitro, that it still interacts with SUMO and at least one protein, Rad22, which is a SUMO-modified target. The Hus5.62 protein is also capable of forming a thioester link with SUMO, but it does not function in sumoylation assays, either in the modification of Rad22 or in SUMO chain formation. When overexpressed in wild-type S. pombe cells, the Hus5.62 protein has a dominant-negative effect on sumoylation.
Rho family GTPase Rnd2 interacts and co-localizes with MgcRacGAP in male germ cells
Authors: NAUD Nathalie , TOURÉ Aminata , LIU Jianfeng , PINEAU Charles , MORIN Laurence , DORSEUIL Olivier , ESCALIER Denise , CHARDIN Pierre , GACON Gérard
Published: May 2003

The male-germ-cell Rac GTPase-activating protein gene (MgcRacGAP) was initially described as a human RhoGAP gene highly expressed in male germ cells at spermatocyte stage, but exhibits significant levels of expression in most cell types. In somatic cells, MgcRacGAP protein was found to both concentrate in the midzone/midbody and be required for cytokinesis. As a RhoGAP, MgcRacGAP has been proposed to down-regulate RhoA, which is localized to the cleavage furrow and midbody during cytokinesis. Due to embryonic lethality in MgcRacGAP-null mutant mice and to the lack of an in vitro model of spermatogenesis, nothing is known regarding the role and mode of action of MgcRacGAP in male germ cells. We have analysed the expression, subcellular localization and molecular interactions of MgcRacGAP in male germ cells. Whereas MgcRacGAP was found only in spermatocytes and early spermatids, the widespread RhoGTPases RhoA, Rac1 and Cdc42 (which are, to various extents, in vitro substrates for MgcRacGAP activity) were, surprisingly, not detected at these stages. In contrast, Rnd2, a Rho family GTPase-deficient G-protein was found to be co-expressed with MgcRacGAP in spermatocytes and spermatids. MgcRacGAP was detected in the midzone of meiotic cells, but also, unexpectedly, in the Golgi-derived pro-acrosomal vesicle, co-localizing with Rnd2. In addition, a stable Rnd2–MgcRacGAP molecular complex could be evidenced by glutathione S-transferase pull-down and co-immunoprecipitation experiments. We conclude that Rnd2 is a probable physiological partner of MgcRacGAP in male germ cells and we propose that MgcRacGAP, and, quite possibly, other RhoGAPs, may participate in signalling pathways involving Rnd family proteins.
Increased expression of the lipocalin 24p3 as an apoptotic mechanism for MK886
Authors: TONG Zhimin , WU Xuli , KEHRER James P.
Published: May 2003

MK886, a strong proapoptotic agent, is an inhibitor of 5-lipoxygenase (LOX) through binding to the 5-LOX-activating protein (FLAP). Although MK886-induced apoptosis is through a FLAP-independent pathway, the precise mechanisms are not understood. In the present study, a possible role of 24p3, a lipocalin, in MK886-induced apoptosis was investigated. Exposure of murine prolymphoid progenitor cells (FL5.12) to 20 μM MK886 for 16 h dramatically increased 24p3 mRNA and protein expression. Induction could also be achieved with another FLAP inhibitor, MK591. The induction of 24p3 by MK886 was dose- and time-dependent. The up-regulated 24p3 mRNA expression by MK886 was enhanced a further 3.1-fold by WY14643, an activator of peroxisome-proliferator-activated receptor α, whereas ciglitazone, an activator of peroxisome-proliferator-activated receptor γ attenuated the MK886-induced 24p3 expression by more than 50%. Neither WY14643 nor ciglitazone alone had any effect on the expression of 24p3. The induction of 24p3 by MK886 was dependent on the synthesis of new protein(s), since cycloheximide, an inhibitor of protein synthesis, prevented this effect. In all cases, including the inhibition of MK886-induced 24p3 protein expression by stable transfection with antisense cDNA of 24p3, the extent of apoptosis closely paralleled 24p3 levels. Apoptosis induced by MK886, or enhanced by WY14643, was accompanied by the cleavage and activation of caspase-3. The overexpression of bcl-2 or bcl-xL in FL5.12 cells inhibited apoptosis induced by MK886 as well as the enhancement of apoptosis by WY14643. Thus 24p3 is an MK886-inducible gene and may play an important role in MK886-induced apoptosis.
Mannose metabolism is required for mycobacterial growth
Authors: PATTERSON John H. , WALLER Ross F. , JEEVARAJAH Dharshini , BILLMAN-JACOBE Helen , McCONVILLE Malcolm J.
Published: May 2003

Mycobacteria are the causative agents of tuberculosis and several other significant diseases in humans. All species of mycobacteria synthesize abundant cell-wall mannolipids (phosphatidylinositol mannosides, lipoarabinomannan), a cytoplasmic methylmannose polysaccharide and O-mannosylated glycoproteins. To investigate whether these molecules are essential for mycobacterial growth, we have generated a Mycobacterium smegmatis mannose auxotroph by targeted deletion of the gene encoding phosphomannose isomerase (PMI). The PMI deletion mutant displayed a mild hyperseptation phenotype, but grew normally in media containing an exogenous source of mannose. When this mutant was suspended in media without mannose, ongoing synthesis of both the mannolipids and methylmannose polysaccharides was halted and the hyperseptation phenotype became more pronounced. These changes preceded a dramatic loss of viability after 10 h in mannose-free media. Mannose starvation did not lead to detectable changes in cell-wall ultrastructure or permeability to hydrophobic drugs, or to changes in the rate of biosynthesis of other plasma-membrane or wall-associated phospholipids. These results show that mannose metabolism is required for growth of M. smegmatis and that one or more mannose-containing molecules may play a role in regulating septation and cell division in these bacteria.
Phosphorylation induces subtle structural changes in SpoIIAA, a key regulator of sporulation
Authors: CLARKSON Joanna , CAMPBELL Iain D. , YUDKIN Michael D.
Published: May 2003

The phosphorylation state of SpoIIAA is a key factor in the regulation of sporulation in Bacillus subtilis. Previous crystallographic studies had led to the conclusion that phosphorylation alters the binding affinity of SpoIIAA for its partner proteins solely through the additional charge and bulk of the phosphoryl group: small structural changes observed elsewhere in the protein were considered to be random fluctuations rather than the result of phosphorylation. The results presented in the present paper show that NMR studies detect the same subtle structural changes in solution as those seen in the crystal, strongly implying that they are the direct result of phosphorylation. These subtle structural changes are similar to those that occur in a non-phosphorylated mutant that is defective in binding to one of its partner proteins. We propose that the structural changes which occur in SpoIIAA on phosphorylation act in concert with the phosphoryl group to alter its binding properties.
The role of an evolutionarily conserved cis-proline in the thioredoxin-like domain of human class Alpha glutathione transferase A1-1
Authors: NATHANIEL Chris , WALLACE Louise A. , BURKE Jonathan , DIRR Heini W.
Published: May 2003

The thioredoxin-like fold has a βαβαββα topology, and most proteins/domains with this fold have a topologically conserved cis-proline residue at the N-terminus of β-strand 3. This residue plays an important role in the catalytic function and stability of thioredoxin-like proteins, but is reported not to contribute towards the stability of glutathione S-transferases (GSTs) [Allocati, Casalone, Masulli, Caccarelli, Carletti, Parker and Di Ilio (1999) FEBS Lett. 445, 347–350]. In order to further address the role of the cis-proline in the structure, function and stability of GSTs, cis-Pro-56 in human GST (hGST) A1-1 was replaced with a glycine, and the properties of the P56G mutant were compared with those of the wild-type protein. Not only was the catalytic function of the mutant dramatically reduced, so was its conformational stability, as indicated by equilibrium unfolding and unfolding kinetics experiments with urea as denaturant. These findings are discussed in the context of other thioredoxin-like proteins.