bluuebunny/crossref_metadata_2025 huggingface dataset:
On the origins of medfly invasion and expansion in Australia
Authors: BONIZZONI M. , GUGLIELMINO C. R. , SMALLRIDGE C. J. , GOMULSKI M. , MALACRIDA A. R. , GASPERI G.
Published: November 2004

As a result of their rapid expansion and large larval host range, true fruit flies are among the world's most important agricultural pest species. Among them, Ceratitis capitata has become a model organism for studies on colonization and invasion processes. The genetic aspects of the medfly invasion process have already been analysed throughout its range, with the exception of Australia. Bioinvasion into Australia is an old event: medfly were first captured in Australia in 1895, near Perth. After briefly appearing in Tasmania and the eastern states of mainland Australia, medfly had disappeared from these areas by the 1940s. Currently, they are confined to the western coastal region. South Australia seems to be protected from medfly infestations both by the presence of an inhospitable barrier separating it from the west and by the limited number of transport routes. However, numerous medfly outbreaks have occurred since 1946, mainly near Adelaide. Allele frequency data at 10 simple sequence repeat loci were used to study the genetic structure of Australian medflies, to infer the historical pattern of invasion and the origin of the recent outbreaks. The combination of phylogeographical analysis and Bayesian tests showed that colonization of Australia was a secondary colonization event from the Mediterranean basin and that Australian medflies were unlikely to be the source for the initial Hawaiian invasion. Within Australia, the Perth area acted as the core range and was the source for medfly bioinvasion in both Western and South Australia. Incipient differentiation, as a result of habitat fragmentation, was detected in some localized areas at the periphery of the core range.
Impacts of gene flow and logging history on the local genetic structure of a scattered tree species,Sorbus torminalisL. Crantz
Authors: ODDOU‐MURATORIO SYLVIE , DEMESURE‐MUSCH BRIGITTE , PÉLISSIER RAPHAËL , GOUYON PIERRE‐HENRI
Published: November 2004

Sorbus torminalisL. Crantz is a colonizing tree species usually found at low density in managed European forests. Using six microsatellite markers, we investigated spatial and temporal patterns of genetic structure within a 472‐ha population of 185 individuals to infer processes shaping the distribution of genetic diversity. Only eight young stems were found to be the result of vegetative reproduction. Despite high levels of gene flow (standard deviation of gene dispersal = 360 m), marked patterns of isolation by distance were detected, associated with an aggregated distribution of individuals in ∼100‐m patches. This spatial structure of both genes and individuals is likely to result from patterns of seedling recruitment combined with low tree density. Our results suggest that landscape factors and logging cycles markedly shape the distribution of favourable sites for seedling establishment, which are then colonized by sibling cohorts as a result of joint seed transportation by frugivores. These combined genetic and demographic processes result in similar genetic structure both within and among logging units. However, conversion to high forest may enhance genetic structuring.
Long‐distance gene flow and cross‐Andean dispersal of lowland rainforest bees (Apidae: Euglossini) revealed by comparative mitochondrial DNA phylogeography
Authors: DICK CHRISTOPHER W. , ROUBIK DAVID W. , GRUBER KARL F. , BERMINGHAM ELDREDGE
Published: November 2004

Euglossine bees (Apidae; Euglossini) exclusively pollinate hundreds of orchid species and comprise up to 25% of bee species richness in neotropical rainforests. As one of the first studies of comparative phylogeography in a neotropical insect group, we performed a mitochondrial DNA (mtDNA)‐based analysis of 14 euglossine species represented by populations sampled across the Andes and/or across the Amazon basin. The mtDNA divergences within species were consistently low; across the 12 monophyletic species the mean intraspecific divergence among haplotypes was 0.9% (range of means, 0–1.9%). The cytochrome oxidase 1 (CO1) divergence among populations separated by the Andes (N = 11 species) averaged 1.1% (range 0.0–2.0%). The mtDNA CO1 data set displayed homogeneous rates of nucleotide substitution, permitting us to infer dispersal across the cordillera long after the final Andean uplift based on arthropod molecular clocks of 1.2–1.5% divergence per million years. Gene flow across the 3000‐km breadth of the Amazon basin was inferred from identical cross‐Amazon haplotypes found in five species. Although mtDNA haplotypes for 12 of the 14 euglossine species were monophyletic, a reticulate CO1 phylogeny was recovered in Euglossa cognata and E. mixta, suggesting large ancestral populations and recent speciation. Reference to closely related outgroups suggested recent speciation for the majority of species. Phylogeographical structure across a broad spatial scale is weaker in euglossine bees than in any neotropical group previously examined, and may derive from a combination of Quaternary speciation, population expansion and/or long‐distance gene flow.
Testing geographical pathways of speciation in a recent island radiation
Authors: MENDELSON TAMRA C. , SIEGEL ALEX M. , SHAW KERRY L.
Published: November 2004

Determining the mode, or geographical context, of speciation is a critical first step to understanding the evolutionary mechanisms that cause new species to arise. In this study, we estimated phylogenetic relationships in thecerasinaspecies group of the Hawaiian cricket genusLaupala(Orthoptera: Gryllidae) to test competing phylogeographical hypotheses and thus infer the mode of speciation. A previous phylogenetic result based on nuclear sequence data suggested that populations ofL. cerasinaon the Big Island of Hawaii are the result of two independent colonizations from Maui, implying parallel speciation and convergent song evolution, and contradicting systematic hypotheses based on behavioural and morphological data. We used amplified fragment length polymorphisms to investigate further the relationships among species and populations in thecerasinaspecies group. Results of these analyses provide a robust estimate of phylogenetic relationships and support the phylogeographical history indicated by behavioural and morphological data.
Presence of D110 antigen expressing immunocompetent cells in glioblastoma associates with prolonged survival
Authors: Preusser M. , Ströbel T. , Birner P. , Marosi C. , Dieckmann K. , Rössler K. , Budka H. , Lassmann H. , Hainfellner J. A.
Published: November 2004

Monoclonal antibody D110 has recently been described as a novel marker of hypoxic tissue damage, reacting with a so far unknown antigen with preferential expression in the central nervous system. The aim of the study was to investigate D110 immunoreactivity in glioblastoma, its association with the expression of hypoxia‐related proteins and its impact on patient outcome. A total of 114 consecutive adult patients who underwent first operation of primary glioblastoma were included in this study. We evaluated D110 immunoreactivity qualitatively and semi‐quantitatively and correlated it with expression of hypoxia inducible factor 1 alpha (HIF‐1α), expression of vascular endothelial growth factor (VEGF), and with patient survival using univariate and multivariate statistical analysis. We observed D110 immunolabelling in 85.1% of the cases. D110 immunoreactivity was detectable in infiltrating HLA‐DR and CD68 expressing cells, most likely microglial cells or haematogenous cells of monocyte/macrophage lineage. In the peripheral lymphoreticular system, immunohistochemistry disclosed selective D110 labelling of Langerhans cells and of dendritic cells of the thymic medulla. Univariate statistical analysis revealed significantly longer survival of patients whose glioblastomas contained D110 immunoreactive infiltrating cells. There was no association between presence of D110 immunoreactive cells and expression of HIF‐1α and VEGF. We conclude that D110 immunoreactivity in glioblastoma does not seem to be related to tissue hypoxia. D110 identifies immunocompetent cells, which positively influence survival of glioblastoma patients.
Depletion of MAP2 expression and laminar cytoarchitectonic changes in dorsolateral prefrontal cortex in adult autistic individuals
Authors: Mukaetova‐Ladinska E. B. , Arnold H. , Jaros E. , Perry R. , Perry E.
Published: November 2004

The neuropathological substrates underlying the characteristic clinical phenotype of autism are unknown. Neuroimaging studies have identified a decrease in task‐related activation in the dorsolateral prefrontal cortex in autism. In the current study, we have analysed the dorsolateral prefrontal cortex in two adult individuals with a clinical diagnosis of autism, using Nissl staining and MAP2 immunohistochemistry. There was unchanged density of both neuronal and glial cell pools, although the autistic individuals had ill‐defined neocortical cellular layers, substantially depleted MAP2 neuronal expression, and reduced dendrite numbers. Further studies on a larger number of individuals with autism are needed to establish the clinical relevance of the described changes, especially to determine whether the loss of dendritic markers is age associated or disease specific.
Tau aggregates are abnormally phosphorylated in inclusion body myositis and have an immunoelectrophoretic profile distinct from other tauopathies
Authors: Maurage C.‐A. , Bussière T. , Sergeant N. , Ghesteem A. , Figarella‐Branger D. , Ruchoux M.‐M. , Pellissier J.‐F. , Delacourte A.
Published: November 2004

Sporadic inclusion body myositis (s‐IBM) is the most frequent progressive acquired inflammatory myopathy in people older than 50 years. Abnormal aggregates of ‘Alzheimer's proteins’, including tau proteins, have been previously demonstrated in s‐IBM. In the present study, we have investigated by immunohistochemistry and immunoblotting analysis the presence of tau proteins in muscle biopsy samples from patients with s‐IBM and other myopathies with rimmed vacuoles, using newly developed antibodies raised against tau protein epitopes found in Alzheimer's disease brain. Tau immunoreactivity was shown in rimmed vacuoles or inclusions, preferentially with antibodies directed against phosphorylated carboxy‐terminal epitopes of tau proteins. Cytoplasmic reactivity was also demonstrated in atrophic, nonvacuolated fibres, as well as in non‐necrotic fibres invaded by inflammatory cells. Abnormally phosphorylated tau aggregates were also  found  in  other  compartments  of  the  muscle  fibre  in s‐IBM and other myopathies. Tau immunoblotting showed an electrophorectic profile of a doublet within the range of 60–62 kDa isovariants, which was different from tauopathies of the central nervous system. Finally, the unique pattern of immunoreactivity of s‐IBM samples towards anti‐tau antibodies is a new clue to a possible distinct subclass of peripheral tauopathy, different from the tauopathies of the central nervous system.
Sharing of polyglutamine localization by the neuronal nucleus and cytoplasm in CAG‐repeat diseases
Authors: Yamada M. , Tan C.‐F. , Inenaga C. , Tsuji S. , Takahashi H.
Published: November 2004

The expansion of a trinucleotide cytosine adenine and guanine (CAG) repeat that codes for polyglutamine is a common gene mutation in the family of hereditary neurodegenerative diseases that includes Machado‐Joseph disease (MJD) and dentatorubral‐pallidoluysian atrophy (DRPLA). The presence of ubiquitinated neuronal intranuclear inclusions (NIIs) has been recognized as a neuropathological hallmark of these diseases, although the significance of NIIs in the pathogenesis remains a matter of controversy. In a previous study of DRPLA, we proposed that intranuclear diffuse accumulation of mutant proteins is another pathological characteristic of neurones, and that the variable prevalence of this characteristic may be relevant to the variation of clinical symptoms in patients with different repeat sizes. Recently, we also disclosed that polyglutamine tracts are localized in a subset of lysosomes in affected neurones. The present immunohistochemical study of autopsied MJD and DRPLA brains shows that the nucleus and cytoplasm of affected neurones share the subcellular distribution of expanded polyglutamine tracts, the pattern of distribution being specific to each diseased brain. The results suggest that in CAG‐repeat diseases, mutant proteins are involved in both the ubiquitin/proteasome and endosomal/lysosomal pathways for protein degradation in different intraneuronal compartments, where their accumulation may exert distinct influences on neuronal physiology.
The extent of axonal loss in the long tracts in hereditary spastic paraplegia
Authors: DeLuca G. C. , Ebers G. C. , Esiri M. M.
Published: November 2004

Hereditary spastic paraplegia (HSP) comprises a group of inherited neurodegenerative disorders with the shared characteristics of progressive weakness and spasticity predominantly affecting the lower limbs. Limited pathological accounts have described a ‘dying back’ axonal degeneration in this disease. However, the distribution and extent of axonal loss has not been elucidated in a quantitative way. We have studied post‐mortem material from six HSP patients and 32 controls in detail. The population of axons was examined quantitatively in the corticospinal tracts from the medulla to the lumbar spinal cord and the sensory tracts from the lumbar to upper cervical spinal cord. Myelin and axon‐stained sections were employed to estimate the notional area and axonal density, respectively, of both tracts. Our results indicate that in the corticospinal tracts there is a significant reduction in area and axonal density at all levels investigated in HSP compared to controls. In the corticospinal tracts, the ratio of medulla and lumbar total axonal number was significantly greater in HSP cases compared to controls suggesting more pronounced axonal loss in the distal neuraxis in HSP than in controls. The sensory tracts in HSP, in contrast, showed a significant reduction in area and axonal density only in the upper regions of the spinal cord. Similar to the corticospinal tracts, the ratio of lumbar and upper cervical cord total axonal number in the sensory tracts was increased in HSP cases compared to controls. These findings are consistent with a length‐dependent ‘dying back’ axonopathy. Nerve fibre loss was not size‐selective with both small and large diameter fibres affected. In HSP, axonal loss is widespread and symmetrical and its extent tract‐specific. The characterization of the nature of axonal loss in HSP, where this is a primary phenomenon, may help the interpretation of axonal loss in conditions such as multiple sclerosis where the sequence of events is less clear.
DEET mosquito repellent provides personal protection against malaria: a household randomized trial in an Afghan refugee camp in Pakistan
Authors: Rowland Mark , Downey Gerald , Rab Abdur , Freeman Tim , Mohammad Nasir , Rehman Hamid , Durrani Naeem , Reyburn Hugh , Curtis Chris , Lines Jo , Fayaz Mohammad
Published: November 2004

Synthetic repellents based on di‐ethyl 3‐methyl benzamide (DEET) are a popular method of obtaining protection from mosquitoes and yet clear evidence for a protective effect against malaria has hitherto never been convincingly demonstrated. A household randomized trial was undertaken among a study population of 127 families (25%) in an Afghan refugee village in Pakistan to compare the efficacy of repellent soap (MosbarTM containing 20% DEET and 0.5% permethrin) vs. a placebo lotion. Cases of falciparum and vivax malaria were detected by passive case detection at the camp's clinic. At the end of the 6 month trial 3.7% (23 of 618) of individuals in the Mosbar group had presented with one or more episodes of falciparum malaria compared with 8.9% (47 of 530) of the placebo group (odds ratio 0.44, 95% CI 0.25–0.76). 16.7% of the Mosbar group (103 of 618) presented with vivax malaria compared with 11.7% (62 of 530) of the placebo group, and thus no effect was shown against vivax malaria (odds ratio 1.29, 95% CI 0.86–1.94). A considerable proportion of individuals (22%) had presented with vivax malaria during the 7 months leading up to the trial and thus any intervention effect would be partially masked by relapsed infections. The distribution of mosquitoes among households was broadly similar between Mosbar and placebo groups. The repellent was popularly received and very few side‐effects were reported. There is a case for giving repellents more prominence in public health as a preventive measure in regions where vectors bite in the early evening or in emergency situations such as epidemics or newly established refugee camps.
Short Communication: Prevalence of mutations associated with resistance to atovaquone and to the antifolate effect of proguanil in Plasmodium falciparum isolates from northern Ghana
Authors: Muehlen Marion , Schreiber Jonas , Ehrhardt Stephan , Otchwemah Rowland , Jelinek Tomas , Bienzle Ulrich , Mockenhaupt Frank P.
Published: November 2004

Atovaquone‐proguanil has recently been introduced for the treatment and prophylaxis of malaria. However, resistance of Plasmodium falciparum is increasingly reported. We assessed P. falciparum polymorphisms associated with resistance to atovaquone (cytochrome b, cytb) and to cycloguanil, the active compound of proguanil (dihydrofolate reductase, dhfr) in 100 isolates from northern Ghana. None of these exhibited cytb codon 268 mutations. Moreover, no dhfr V16A, S108T or I164L mutations linked with cycloguanil resistance were detected. However, dhfr triple mutants (S108N‐I51L‐C59R) conferring resistance to proguanil and sulphadoxine‐pyrimethamine were seen in 51% of the isolates. In northern Ghana, P. falciparum cytb codon 268 mutations associated with atovaquone resistance are absent. Although proguanil appears to act synergistically with atovaquone in a way different from its antifolate property, the abundance of dhfr polymorphisms will likely compromise the prevention of dissemination of atovaquone‐resistant parasites once emerged.
Split‐drug regimens for the treatment of patients with sputum smear‐positive pulmonary tuberculosis – a unique approach
Authors:   Tuberculosis Research Centre (Indian Council of Medical Research), Chennai, India
Published: November 2004

Objective  To evaluate the efficacy of split‐drug regimens for treatment of patients with sputum smear‐positive pulmonary tuberculosis in south India.

Design  Randomized controlled clinical trial where eligible patients were randomly allocated to: (i) 2RE3HZ3(alt)/4RH2 (split I): rifampicin plus ethambutol given on one day and isoniazid plus pyrazinamide the next day for first 2 months followed by rifampicin plus isoniazid twice weekly for 4 months, or (ii) 3RE3HZ3(alt)/3RH2 (split II): similar to regimen 1, except duration was 3 months in each phase, or (iii) 2REHZ3/4RH2 (control): rifampicin, isoniazid, ethambutol and pyrazinamide, given thrice weekly for 2 months followed by isoniazid and rifampicin twice weekly for 4 months. All patients were followed up clinically and bacteriologically every month up to 2 years and every 6 months for up to 5 years.

Results  A favourable response (cultures negative for Mycobacterium tuberculosis during the last 2 months of treatment) was observed in 91% of 407 patients in split I, 94% of 415 in split II and 89% of 418 in the control regimen. Ninety‐one per cent of 370 patients in split I, 93% of 389 in split II and 90% of 370 in control regimens had quiescent disease at the end of 60 months. Gastrointestinal symptoms were more frequent under the control regimen (P = 0.01).

Conclusion  Split‐drug regimens were as effective as the control regimen in terms of favourable response at the end of treatment and quiescent disease at 5 years, and caused fewer gastrointestinal side‐effects.
A community study using specified and unspecified scenarios to investigate men's views about PSA screening
Authors: Gattellari Melina , Ward Jeanette E.
Published: November 2004

Objective  We explored the influence of different but factual scenarios about prostate‐specific antigen (PSA) screening on men's interest in having PSA screening to detect early prostate cancer.

Design  Cross‐sectional, representative community survey.

Setting and participants  A total of 514 men (89% response fraction) aged 50–70 years randomly selected from a telephone directory database in Sydney, Australia.

Main variables studied  Demographic, health and psychological variables.

Main outcome variables  Interest in undergoing screening in response to five unspecified scenarios and, elsewhere in our interview, a specified scenario in which PSA screening was mentioned explicitly.

Results  When presented with a scenario describing a lack of evidence underpinning the efficacy of screening for an unspecified cancer, 61.2% of men reported that they ‘probably’ or ‘definitely’ wanted to undergo screening for an unspecified cancer. Similar proportions reported that they ‘probably’ or ‘definitely’ wanted to undergo screening even at the risk of unmasking indolent cancer (60.9%) or without expert consensus about the value of screening (62.8%). Greatest interest in screening was elicited in that scenario describing life‐time risk of dying from prostate cancer (72.6%) (P < 0.001). Significantly fewer indicated they would ‘probably’ or ‘definitely’ want to undergo screening for a cancer for which there was uncertainty about treatment efficacy and known side‐effects (46.1%) (P < 0.001). Increasing age was a consistent predictor of positive interest in screening. When asked later in our survey specifically about PSA screening, 68.1%‘probably’ or definitely’ wanted PSA screening.

Conclusion  Public health policy makers need to ensure that men are provided with the scope of medical evidence germane to prostate cancer screening and treatment, thereby potentially improving prostate cancer screening decisions.
Cardiovascular disease in renal allograft recipients is associated with elevated sialic acid or markers of inflammation
Authors: Bakri Rashed S , Afzali Behdad , Covic Adrian , Sriskantharan Rajaventhan , Bharma‐Ariza Paul , Park Won‐Ho , Sriharan Mohanaluxmi , Dalton Neil , Wierzbicki Anthony S , Crook Martin A , Goldsmith David JA
Published: November 2004

Abstract:  Background:  Cardiovascular disease (CVD) is more common in patients with renal failure, even after renal transplantation. We wished to examine the relationship between markers of inflammation and CVD in stable renal transplant patients.

Methods:  Ninety stable renal transplant outpatients with no recent illnesses or rejection were invited for study. Blood was drawn for a variety of inflammatory markers including total plasma sialic acid (SA) levels.

Results:  Patients with CVD were significantly older than patients without (54 ± 12 vs. 42 ± 14 yr, p < 0.01) and had significantly lower total cholesterol (4.5 ± 1.6 vs. 5.1 ± 1.0 mmol/L, p < 0.01). Time from transplantation, present creatinine and blood pressure, smoking history were similar in both groups. Patients with CVD had significantly higher levels of SA (89.2 ± 22.3 vs. 77.4 ± 13.9 mg/dL, p = 0.01); fibrinogen [4.6 (2.2–6.7) vs. 3.6(1.9–5.7) g/L; p = 0.05); and C‐reactive protein (CRP) [2.2 (1.5–8.0) vs 1.5 (0.7–3.0) μg/dL] than those without CVD. A logistic multiple linear regression analysis of the data with CVD as the dependent variable, and all the other parameters as independent variables, showed significant associations (F = 16.9; p < 0.001) with diastolic blood pressure (β = 5.6; p = 0.02) and CRP (β = 4.4; p = 0.04).

Conclusions:  This study suggests that inflammation is associated with a higher prevalence of cardiovascular disease in patients with renal allografts. The measurement of sialic acid as a risk factor may be superior to that of CRP in this group as its concentration is independent of renal function.
Cyclosporin A prodrugs: design, synthesis and biophysical properties
Authors: Hamel A.R. , Hubler F. , Carrupt A. , Wenger R.M. , Mutter M.
Published: November 2004

Abstract:  The cyclic undecapeptide cyclosporin A (CsA) has a remarkable spectrum of diverse biological activities, including anti‐inflammatory, antifungal, antiparasitic as well as immunosuppressive activities. However, the low water solubility of this drug is a serious problem causing undesirable pharmacological properties such as erratic oral absorption. In order to overcome this problem, the design and synthesis of water‐soluble prodrugs of CsA are described. Using the OH‐MeBmt‐1‐group as attachment site, we investigate dipeptide systems exhibiting differential tendencies for intramolecular cyclization [diketopiperazine (DKP) formation] for tailoring the chemoreversible release of the parent CsA. In modulating the chemical and structural features of the dipeptide esters (N‐alkylation, side chains, C‐terminal Pro), we find conversion rates at physiological conditions ranging from minutes to several days. Together with their thermodynamic stability in the solid state and strongly enhanced solubility in water, these chemoreversible CsA prodrugs represent versatile candidates for therapeutical use.
Synthesis of symmetrically and asymmetrically branched pegylating reagents
Authors: Felix A.M. , Bandaranayake R.M.
Published: November 2004

Abstract:  A solution‐phase procedure using an orthogonal protection scheme was developed for the synthesis of a novel family of multi‐pegylating reagents. The procedure was exemplified by the synthesis of bis‐ and tris‐pegylating reagents prepared by stepwise insertion of the poly(ethylene glycol) units thereby enabling the preparation of both symmetrical and asymmetrical pegylating reagents. Asymmetrical pegylation and tris‐pegylation of peptides and proteins introduces new variables for use in the optimization of pegylated peptides and proteins. These reagents are ideally suited for conjugation to peptides and proteins as they possess a required functional group and will be useful intermediates for the synthesis of a new generation of pegylated products. Tris‐pegylation can also provide more effective protection from proteolysis by shielding the protein surface from approaching macromolecules. To illustrate this potential, conditions were developed for the successful coupling of the tris‐pegylating reagent to a model pentapeptide.
Structure‐based design, synthesis, and pharmacologic evaluation tf peptide RGS4 inhibitors
Authors: Jin Y. , Zhong H. , Omnaas J.R. , Neubig R.R. , Mosberg H.I.
Published: November 2004

Abstract:  Regulators of G‐protein signaling (RGS) proteins form a multifunctional signaling family. A key role of RGS proteins is binding to the G‐protein Gα‐subunit and acting as GTPase‐activating proteins (GAPs), thereby rapidly terminating G protein‐coupled receptor (GPCR) signaling. Using the published RGS4–Giα1 X‐ray structure we have designed and synthesized a series of cyclic peptides, modeled on the Giα Switch I region, that inhibit RGS4 GAP activity. These compounds should prove useful for elucidating RGS‐mediated activity and serve as a starting point for the development of a novel class of therapeutic agent.
Synthesis, conformation, receptor binding and biological activities of monobiotinylated human insulin‐like peptide 3*
Authors: Fu P. , Layfield S. , Ferraro T. , Tomiyama H. , Hutson J. , Otvos L. , Tregear G.W. , Bathgate R.A.D. , Wade J.D.
Published: November 2004

Abstract:  Biotin‐avidin immobilization has been routinely used as a tool to study peptide–receptor and peptide–antibody interactions. Biotinylated peptides can also be employed to localize cells that express the peptides’ receptor, and to analyse ligand‐receptor binding. Insulin‐like peptide 3 (INSL3) is a peptide hormone which contains A‐ and B‐chains connected by two disulphide bonds and plays a role in testicular descent during sexual development. In order to study the interaction of INSL3 with its receptor LGR8, a G protein‐coupled receptor, we chemically synthesized Nα‐mono‐biotinylated human INSL3 (B‐hINSL3) and compared it structurally and biologically with hINSL3. Both peptides exhibited similar, but high, receptor binding affinities on human foetal kidney fibroblast 293T cells transfected human LGR8 based on a competition radioreceptor assay with 33P‐labelled relaxin H2 (B33). The modified B‐hINSL3 showed full biological activity as determined by the stimulation of gubernacular cell proliferation. The labelled B‐hINSL3 contains a higher α‐helix content, and this increased helical structure is accompanied by an increase in ability to stimulate cAMP accumulation in 293T cells expressing LGR8. Our results suggest that the N‐terminal region of the A‐chain is not involved in the interaction of INSL3 with its receptor. However, the introduction of biotin onto the N‐terminus of the A‐chain promoted conformational stability which, in turn, permitted better receptor activation.
In vivo properties of thiol inhibitors of the three vasopeptidases NEP, ACE and ECE are improved by introduction of a 7‐azatryptophan in P2′ position
Authors: Inguimbert N. , Poras H. , Dhotel H. , Beslot F. , Scalbert E. , Bennejean C. , Renard P. , Fournié‐Zaluski M.‐C. , Roques B.‐P.
Published: November 2004

Abstract:  Three zinc metallopeptidases are implicated in the regulation of fluid homeostasis and vascular tone and represent interesting targets for the treatment of chronic heart failure. We have previously reported the synthesis of a triple inhibitor able to simultaneously inhibit neprilysin (NEP, EC 3.4.24.11), angiotensin‐converting enzyme (ACE, EC 3.4.15.1) and endothelin‐converting enzyme (ECE‐1, EC 3.4.24.71) with nanomolar potency towards NEP and ACE and a lesser affinity for ECE. Here, we report the optimization and biological activities of analogs derived from lead compound 1 (2S)‐2‐[(2R)‐2‐((1S)‐5‐bromo‐indan‐1‐yl)‐3‐mercapto‐propionylamino]‐3‐ (1H‐indol‐3‐yl)‐propionic acid by a structural approach. Among several inhibitors, compound 21, (2S)‐2‐[(2R)‐2‐((1S)‐5‐bromo‐indan‐1‐yl)‐3‐mercapto‐propionylamino]‐3‐(1H‐pyrrolo[2,3‐b]pyridin‐3‐yl)‐propionic acid was selected by taking into account its good molecular adaptation with the recently published structures of the three vasopeptidases. This optimization procedure led to an improved pharmacologic activity when compared with 1.
Synthesis of substrates and inhibitors of botulinum neurotoxin type A metalloprotease*
Authors: Sukonpan C. , Oost T. , Goodnough M. , Tepp W. , Johnson E.A. , Rich D.H.
Published: November 2004

Abstract: Botulinum neurotoxin (BoNT) metalloproteases and related proteases are the most selective proteases known. X‐ray crystal structures suggest that the active sites of the native enzymes exist in catalytically incompetent forms that must be activated by substrate binding. In order to characterize the postulated substrate‐induced conformational changes for enzyme activation, we synthesized a series of transition‐state analog inhibitors in which the dipeptide cleavage site is replaced by tetrahedral intermediate analogs within the minimal substrate peptide sequence. In this paper, we report our efforts to design inhibitors of BoNT/A metalloprotease. We confirm that an effective substrate sequence for BoNT/A metalloprotease is a 17‐mer peptide corresponding to residues 187–203 of SNAP‐25. A more stable substrate, Nle202SNAP‐25 [187–203] was synthesized in order to develop an assay for proteolytic activity of BoNT/A metalloprotease that can be used to monitor time‐dependent inhibition.α‐Thiol amide analogs of Gln‐197 were incorporated via solid‐phase peptide synthesis into both 17‐mer minimal peptide substrate sequences. The synthesis, characterization and inhibition kinetics for theα‐thiol amide analogs of holotoxin A substrate are described. These substrate‐derived inhibitors were shown to be submicromolar inhibitors of BoNT/A catalytic activity.
Relative effects of GAL+ and GALlow/− porcine hematopoietic cells on primate platelet aggregation and endothelial cell activation: implications for the induction of mixed hematopoietic chimerism in the pig‐to‐primate model
Authors: Knosalla Christoph , Giovino Maria A. , Harper David , Kaczmarek Elzbieta , Gollackner Bernd , Cooper David K. C. , Robson Simon C.
Published: November 2004

Abstract:  Background:  The induction of porcine hematopoietic cell chimerism in preconditioned baboons has been hampered by the development of thrombotic microangiopathy. As pigs that lack expression of Galα1,3 Gal (Gal) may become available in the near future, we have explored the effects of porcine hematopoietic cells that express low or no Gal (Gallow/−) on baboon platelet aggregation and on human umbilical vein endothelial cell (HUVEC) activation.

Methods:  Porcine mobilized peripheral blood progenitor cells (PBPC; Gal+) and bone marrow mononuclear cells (BM; Gal+ or Gallow/−) were investigated for their potential to (i) induce aggregation of baboon platelets, and (ii) to activate endothelial cells as measured by increased expression of vascular cell adhesion molecule‐1 (VCAM‐1), intercellular adhesion molecule‐1 (ICAM‐1), and E‐selectin on HUVEC. α‐Galactosidase‐treated PBPC were also investigated for their effect on platelet aggregation.

Results:  Gal+ PBPC and Gal+ BM cells (107) induced aggregation of baboon platelets by 42 and 31%, respectively, whereas Gallow/− BM cells did not induce any platelet aggregation. α‐Galactosidase‐treated PBPC induced less platelet aggregation than untreated PBPC. Gal+ PBPC and Gal+ BM cells (107) increased expression of VCAM‐1, ICAM‐1 and E‐selectin on HUVEC, whereas Gallow/− BM cells did not.

Conclusions:  In contrast to Gal+ PBPC or BM, Gallow/− BM cells do not induce aggregation of baboon platelets or activate HUVEC. The induction of tolerance through mixed hematopoietic cell chimerism may be facilitated when α‐galactosyltransferase‐knockout pigs become available.
In vitro and in vivo investigation of a novel monoclonal antibody to plasma cells (W5 mAb)
Authors: Harper D. , Gollackner B. , Xu Y. , Calderhead D. , Ryan D. , Li W. , Chang J. , Wu C. , Moran K. , Latinne D. , Bazin H. , White‐Scharf M. E. , Cooper D. K. C. , Awwad M.
Published: November 2004

Abstract:  Natural antibodies (Abs), predominately anti‐Galα1–3Gal (Gal) Abs, in non‐human primates and human beings present a major hurdle to successful pig‐to‐primate xenotransplantation. Attempts to inhibit anti‐Gal Ab production in naïve baboons using non‐specific immunosuppressive or B cell‐specific reagents have failed. A new rat monoclonal antibody (W5 mAb) has been generated, which binds to all B cells, including memory cells, and to the majority of plasma cells, but not to T cells. It has been tested in vitro and in vivo. By immunoprecipitation, W5 mAb bound a human leukocyte antigen class II (HLA‐DR) determinant. Sorting splenic or bone marrow W5+ cells resulted in a highly enriched anti‐Gal Ab and total immunoglobulin (Ig)‐secretory population. In vivo studies in baboons demonstrated that W5 mAb was safe but, despite the concomitant administration of an anti‐CD154 mAb to inhibit sensitization, anti‐rat Abs were detected within 10 days and inhibited the effect of the W5 mAb. High levels of W5 mAb were able to completely deplete B cells in the blood, but not in lymphoid tissues. Enzyme‐linked spot‐forming assay (ELISPOT) demonstrated that only 50 to 60% of secreting cells (SC) were depleted in the bone marrow. No reduction in the serum levels of anti‐Gal Ab was observed. W5 mAb did not cause complete inhibition of anti‐Gal Ab production, probably as a result of its inability to completely deplete B and plasma cells from all lymphoid compartments.
Clinical nurse specialist care managers’ time commitments in a disease‐management program for bipolar disorder
Authors: Glick Henry A , Kinosian Bruce , McBride Linda , Williford William O , Bauer Mark S ,   the CSP #430 Study Team
Published: November 2004

Objectives:  As part of a cost‐effectiveness analysis for Department of Veterans Affairs Cooperative Studies Program #430, ‘Reducing the Efficacy‐Effectiveness Gap in Bipolar Disorder,’ we conducted a time and motion study to quantify the time psychiatric clinical nurse specialist (CNS) care managers spent providing care for patients.

Methods:  Clinical nurse specialist care managers completed activity logs in which they recorded time spent implementing the Bipolar Disorders Program (BDP) during a 1‐week period in spring, summer, fall and winter over a 1‐year period when caseloads were at steady state. Mean service time was estimated by use of univariate analysis of means and by multivariable regression analysis.

Results:  On average CNS care managers spent 40% of their clinical time in activities that typically are reimbursed (e.g. clinic visits) and spent the remaining 60% of their time in activities that are typically unreimbursed. Total clinic time increased as the number of visits per day increased; however, this increase got smaller with each additional visit per day.

Conclusions:  As with other chronic illness management programs, CNS care managers expend a substantial portion of their clinical effort for the BDP in activities that are typically unreimbursed. Their activities have a fixed component per day as well as a component that systematically varies with the number of visits per day. These findings should be considered when costing out and disseminating psychiatric and other medical chronic illness management programs.
The Effect of Vascular Endothelial Growth Factor on in vitro Embryonic Heart Development in Rats
Authors: Ülger H. , Özdamar S. , Unur E. , Pratten M. K.
Published: November 2004

In vitro effects of vascular endothelial growth factor (VEGF) on heart development and total embryonic growth were investigated in 84 rat embryos (obtained from nine pregnant females) at 9.5 days of gestation that were cultured in whole rat serum (WRS), in <30 kDa + >50 kDa serum fractions [retenate (R)], and in R + VEGF. After 24‐h culture, the embryos from each group were harvested and divided into two groups. One group was analysed morphologically and biochemically to obtain embryo protein content, the second group was serially sectioned and examined by light microscopy. Morphological score, embryo protein content, somite number and crown‐rump length of embryos indicated that embryos cultured in R had significant embryonic retardation, whereas the addition of VEGF to R increased embryonic growth and development. The morphological scores for WRS, R and R + VEGF were 57.7 ± 0.87, 46.6 ± 1.90 and 52.1 ± 0.97, somite numbers were 26.5 ± 0.47, 20.1 ± 0.63 and 24.4 ± 0.46, crown‐rump lengths were 3 ± 0.07, 2.4 ± 0.06 and 2.7 ± 0.06 mm, and embryo protein contents were 160.5 ± 7.41, 98.2 ± 4.81 and 141.1 ± 10.96 μg per embryo, respectively. The results of histological examination of heart development were similar. The hearts of embryos grown in R were unseptated and tubular. The atrioventricular endocardial cushions were incompletely developed. The addition of VEGF to R improved heart development. There were no gross morphological differences in the cardiac development between embryos grown in WRS and R + VEGF. In both groups, development of the muscular interventricular septum had begun. Development of the atrioventricular cushions was also similar in both groups and had caused narrowing of the atrioventricular canals, but the atrial septation was not observed.
Decrease of Nuclear Reactivity to Growth‐regulatory Galectin‐1 in Senescent Human Keratinocytes and Detection of Non‐uniform Staining Profile Alterations upon Prolonged Culture for Galectin‐1 and ‐3
Authors: Chovanec M. , Smetana K. , Dvořánková B. , Plzáková Z. , André S. , Gabius H.‐J.
Published: November 2004

Multipotent stem cells (source for interfollicular epidermis, hairs and sebaceous glands) are localized in the bulge region of the outer root sheath of hair follicles, while stem cells giving rise to interfollicular epidermis reside in its basal. Using the multifunctional lectin galectin‐1 as a marker to localize accessible binding sites in situ as a step to figure out galectin functionality in stem cells, we studied hair follicle‐derived keratinocytes. Specific nuclear binding of galectin‐1 associated with expression of ΔNp63α, a potential marker of epidermal stem cells, was detected. Binding of chimera‐type galectin‐3 to a nuclear site was not found in parallel assays. During the process of ageing in culture when cells acquire properties of senescence, disappearance of the nuclear signal for galectin‐1 binding was accompanied by a similar decrease of nuclear ΔNp63α expression and increased binding of galectin‐3 to the cell membrane, namely in regions of intercellular contacts. Expression of cytokeratin 10, a marker of the terminal differentiation was seen only in a small fraction of the cell population. These data extend the evidence for nuclear sites with galectin‐1 reactivity in squamous epithelial cells, the expression of which is modulated upon senescence. Moreover, the results document the divergence of galectin‐1 and ‐3 on the level of ligand selection in this cell type, underscoring the importance of the technical aspect to employ tissue lectins as probe and to perform a fingerprinting with several markers of the galectin family in parallel.
Immunohistochemical Localization of S‐100 Protein in the Pig Pituitary Gland
Authors: Marettová E. , Maretta M.
Published: November 2004

Immunohistochemical localization of S‐100 protein was studied in anterior, intermediate and posterior lobe of the pig pituitary gland. Two immunopositive cells for S‐100 protein were identified: the folliculo‐stellate cells (FSc) in the glandular lobes and the pituicytes in the neural lobe. In the anterior lobe, immunoreactive folliculo‐stellate cells were scattered among secretory cells. In the area where the secretory cells form strands and follicle‐like groups the positive cells were concentrated in groups. In the intermediate lobe, S‐100 protein‐positive cells were located sparsely among secretory cells and next to secretory follicles and the pituitary cleft. These FSc were more voluminous and displayed fewer cytoplasmic processes. In the neurohypophysis, positive reaction for S‐100 protein was seen in the pituicytes. These cells were distributed singly or concentrated in groups. The distribution, and morphologic characteristics of the FSc in the glandular lobes and the pituicytes in the neural lobe in the pig indicate different origin of both S‐100 protein‐positive cells.
Is the Rhesus Monkey (Macaca mulatta) Comparable to Humans? Histomorphology of the Sphincteric Musculature of the Lower Urinary Tract Including 3D‐reconstruction
Authors: Ganzer R. , Köhler D. , Neuhaus J. , Dorschner W. , Stolzenburg J.‐U.
Published: November 2004

The physiology of the muscle systems of the human lower urinary tract is still not known in detail. To study the functional basics of this complex organ system, experiments are often performed in animal models including rhesus monkeys. To apply the results of animal model studies to the humans, a clear knowledge of the comparative anatomy of both species is necessary. However, detailed comparative studies of the lower urinary tract of the rhesus monkey and the humans are lacking. Accordingly, a detailed study on the sphincteric musculature of the lower urinary tract of the rhesus monkey was performed in order to demonstrate anatomical correspondences and differences between both species. The lower urinary tract anatomy was investigated in 18 male and female rhesus monkeys (Macaca mulatta) by serial sections. Immunohistochemical staining methods were used to differentiate striated and smooth musculature. Three‐dimensional reconstructions were performed in order to demonstrate the topographical anatomy of the different muscle systems. In both man and male rhesus monkeys, a urethral sphincter muscle exists independent of the pelvic floor musculature, with a smooth and a striated muscular part. A urinary diaphragm (diaphragma urogenitale) does neither exist in the rhesus monkey nor in the human. In contrast to women, a striated muscle encircles the urethra and vagina together in the female rhesus monkey. A vesical sphincter muscle, found in the human bladder outlet, does not exist in the rhesus monkey.
PCR‐based identification of Erysiphe pulchra and Phyllactinia guttata from Cornus florida using ITS‐specific primers
Authors: Mmbaga M. T. , Klopfenstein N. B. , Kim M.‐S. , Mmbaga N. C.
Published: November 2004

The internal transcribed spacer (ITS) regions of rDNA and the intervening 5.8S rRNA gene for the powdery mildew fungi Erysiphe (sect. Microsphaera) pulchra and Phyllactinia guttata were amplified using standard polymerase chain reaction (PCR) protocols and the universal primer pairs, ITS1 and ITS4. PCR products for ITS were analysed by electrophoresis in a 1.5% agarose gel and sequenced. The size of the amplified ITS products (approximately 650 bp) were not sufficiently different to allow reliable differentiation of E. pulchra and P. guttata; however, their sequences were distinct. Specific primers for E. pulchra and P. guttata were developed and evaluated for use as diagnostic tools. The diagnostic band size from E. pulchra‐specific primer pair was 568 bp while the P. guttata band was 597 bp; the two primer pairs were highly specific to E. pulchra and P. guttata. Comparison of ITS sequences with information in the GenBank showed a very close similarity between sequences of E. pulchra isolates from Cornus florida in the USA and isolates collected on Cornus kousa in Japan. BLAST analysis of the sequence of the 650‐bp band from P. guttata revealed a close alignment with sequences of P. moricola (92%), P. kakicola (94%), and P. fraxini (92%). The sequence of P. guttata in C. florida also had a 98% identity with P. guttata in Calycanthus occidentalis and 94% identity with P. guttata in Corylus cornuta.
Lysine requirement of kittens given purified diets for maximal growth
Authors: Morris J. G. , Rogers Q. R. , O'Donnell J. A.
Published: November 2004

Three experiments were conducted using a total of 15 male and 15 female weaned specific pathogen‐free kittens given amino acid‐based purified diets containing varying concentrations of lysine (from 4.0 to 11.3 g/kg diet) in a Latin square design of 10 day periods. In experiment 1, the predicted lysine requirement was 7.7 g/kg diet, and in experiments 2 and 3, maximal weight gain occurred at 8.0 g lysine/kg diet. In experiment 3, nitrogen balance was not different for kittens given diets containing 8.0 and 9.0 g lysine/kg. These experiments support a requirement of 8.0 g lysine (free base)/kg diet, in a diet with a calculated metabolizable energy value of 4.7 kcal/g.