bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclin Dependent Kinases and Regulation of the Fission Yeast Cell Cycle
Authors: Nurse P.
Published: October 2004

The cyclin dependent kinases (CDKs), formed by complexes between Cdc2p and the B-cyclins Cig2p and Cdc13p, have a central role in regulating the fission yeast cell cycle and maintaining genomic stability. The CDK Cig2p/Cdc2p controls the onset of S-phase and the CDK Cdc13p/Cdc2p controls the onset of mitosis and ensures that there is only one S-phase in each cell. Cdc13p/Cdc2p can replace Cig2p/Cdc2p for the onset of S-phase, suggesting that the increasing activity of a single CDK during the cell cycle is sufficient to drive a cell in an orderly fashion into S-phase and into mitosis. If S-phase is incomplete, then inhibition of Cdc13p/Cdc2p prevents cells with unreplicated DNA from undergoing a catastrophic entry into mitosis. Control of CDK activity is also important to allow cells to exit the cell cycle and accumulate in G1 in response to nutritional deprivation and the presence of pheromone.
Viroids with Hammerhead Ribozymes: Some Unique Structural and Functional Aspects with Respect to Other Members of the Group
Authors: Flores R. , Navarro J.-A. , Peña M. de la , Navarro B. , Ambrós S. , Vera A.
Published: October 2004

Viroids, subviral pathogens of plants, are composed of a single-stranded circular RNA of 246–399 nucleotides. Within the 27 viroids sequenced, avocado sunblotch, peach latent mosaic and chrysanthemum chlorotic mottle viroids (ASBVd, PLMVd and CChMVd, respectively) can form hammerhead structures in both of their polarity strands. These ribozymes mediate self-cleavage of the oligomeric RNAs generated in the replication through a rolling circle mechanism, whose two other steps are catalyzed by an RNA polymerase and an RNA ligase. ASBVd, and presumably PLMVd and CChMVd, replicate and accumulate in the chloroplast, whereas typical viroids replicate and accumulate in the nucleus. PLMVd and CChMVd do not adopt a rod-like or quasi rod-like secondary structure as typical viroids do but have a highly branched conformation. A pathogenicity determinant has been mapped in a defined region of the CChMVd molecule.
Mutagenesis via Insertional or Restriction Enzyme-Mediated Integration (REMI) as a Tool to Tag Pathogenicity Related Genes in Plant Pathogenic Fungi
Authors: Maier F. J. , Schäfer W.
Published: October 2004

Random insertional mutagenesis is a powerful tool to investigate the molecular basis of most genetically determined processes, for example in pathogenic fungi. An improved version of this method is the insertional mutagenesis via restriction enzyme mediated integration (REMI). Transformation efficiency and mode of vector integration are species dependent and further influenced by vector conformation, restriction enzyme activity, and transformation protocol. An overview is given, covering the mutants and already identified genes obtained after REMI mutagenesis. An outlook describes the future developments in the field.
A Role of Poly (ADP-Ribose) Polymerase in NF- B Transcriptional Activation
Authors: Hassa P. O. , Hottiger M. O.
Published: October 2004

The transcription factor NF-κB plays a critical role in immune and inflammatory responses. Here we show that poly (ADP ribose) polymerase (PARP) is required for specific NF-κB transcriptional activationin vivo. The activation of the HIV-LTR promoter and an NF-κBdependent artificial promoter was drastically reduced in PARP (_/_) cells, independently of the signaling pathway through which NF-bB was induced. Furthermore NF-κB-dependent gene activation was restoredin vivoby the expression of PARP in PARP (_/_) cells. Finally, we show that both NF-κB and PARP formed a stable immunoprecipitable nuclear complex. This interaction did not need DNA binding. Our results suggest that PARP is an important cofactor in the activation cascade of NF-κB-dependent target genes.
The Role of Protein Phosphatase 2A Catalytic Subunit Cα in Embryogenesis: Evidence from Sequence Analysis and Localization Studies
Authors: Götz Jürgen , Kues Wilfried
Published: October 2004

Protein phosphatase 2A (PP2A) constitutes one of the major families of protein serine/threonine phosphatases found in all eukaryotic cells. PP2A holoenzymes are composed of a catalytic subunit complexed with a structural regulatory subunit of 65 kDa. These core subunits associate with regulatory subunits of various sizes to form different heterotrimers which have been purified and evaluated with regard to substrate specificity. In fully differentiated tissues PP2A expression levels are highest in the brain, however, relatively little is known about expression in the developing embryo.

In order to determine the composition of PP2A catalytic subunits in the mouse, cDNAs were cloned and the genomic organization of PP2A Cα was determined.

By a gene targeting approach in the mouse, we have previously shown that the absence of the major catalytic subunit of PP2A, Cα, resulted in embryonic lethality around embryonic day E6.5. No mesoderm was formed which implied that PP2A plays a crucial role in gastrulation.

Here, we extended our studies and analyzed wildtype embryos for Cα expression at subsequent stages of development. After gastrulation is completed, we find high expression of Cα restricted to the neural folds, which suggests that PP2A plays an additional pivotal role in neurulation.
A High Affinity Binding Site for the Polypyrimidine Tract Binding Protein (PTB) Is Located in the 5'-Untranslated Region of the Rat Proteinase α1-Inhibitor 3 Variant I Gene
Authors: Sickinger Stefan , Schweizer Michael
Published: October 2004

As a first step towards understanding the mechanism underlying the differential gene expression of the two variants of the rat proteinase-inhibitor α1-Inhibitor 3 (α1I3) corresponding genomic clones were isolated. The 100% similarity between the sequence of one genomic clone and that of the α1-I3 variant I cDNA strongly suggested that its 5′-sequence represented the upstream region of the corresponding gene. Several putativecis-regulatory elements were identified as well as a polypyrimidine tract located between the transcription start site of the α1-I3 variant I mRNA and the AUG codon. The polypyrimidine tract functions as a positivecis-element in a heterologous promoter. By electrophoretic mobility shift assays (EMSA) we have shown that a GST (glutathione S-transferase) fusion of the rat polypyrimidine tract binding protein (PTB) has a high affinity for the pyrimidine-rich sense strand but not for the complementary sequence of the 5′-untranslated region of the α1-I3 variant I gene.
The Anti-Estrogen Hydroxytamoxifen Is a Potent Antagonist in a Novel Yeast System
Authors: Liu Jia Wei , Jeannin Elisabeth , Picard Didier
Published: October 2004

The budding yeastSaccharomyces cerevisiaehas been used extensively as a biological ‘test tube’ to study the regulation of the human estrogen receptor (ER) α. However, anti-estrogens, which are of great importance as therapeutic agents and research tools, fail to antagonize the activation by estrogen in yeast. Here, we have surveyed the antagonistic potential of five different anti-estrogens of diverse chemical nature. While they all act as agonists for wild-type ERα we have established a novel yeast assay system for anti-estrogens, in which at least the commonly used anti-estrogen hydroxytamoxifen is a potent antagonist.
Standardization and Clinical Management of Lipoprotein(a) Measurements
Authors: Lippi Giuseppe , Guidi Giancesare
Published: October 2004

The present article proposes personal suggestions to improve determinations and clinical interpretation of results of lipoprotein(a) assays. Methods and procedures for sampling and quantification of the various isoforms of lipoprotein(a) in serum, plasma and urine are reviewed with the aim of improving the reliability and reproducibility of results and reinforcing the clinical utility of lipoprotein(a) measurements.
Neopterin Plasma Concentrations Predict the Course of Severe Acute Pancreatitis
Authors: Kaufmann Peter , Tilz Gernot P. , Demel Ulrike , Wachter Helmut , Kreijs Günter J. , Fuchs Dietmar
Published: October 2004

In a prospective, descriptive study in 25 patients with acute pancreatitis neopterin plasma concentrations were found to be associated with the severity of the disease, which was assessed using weights of the worst 17 physiological abnormalities of the APACHE-III score over a 24 h period after hospital admission. Neopterin concentrations were higher in severe pancreatitis (n = 10) compared to mild disease, and there existed a positive exponential correlation between neopterin and the Acute Physiology Score (r = 0.66). Higher neopterin concentrations were associated with the development of multiple organ failure (p = 0.012) and death (p = 0.019). At a cut-off concentration of 12 nmol/l the sensitivity (80 %) and specificity (100 %) of neopterin for the discrimination between mild and severe clinical course of pancreatitis was more accurate than C-reactive protein at a risk threshold of 1.2 g/l (70 % and 87 %). Development of pancreatic necrosis was associated with higher neopterin concentrations than edematous pancreatitis (p < 0.001).
Biological Variation and Quantification of Health: The Emergence of the Concept of Normality
Authors: Büttner Johannes
Published: October 2004

Historical research on the concept of normality, its roots and its development show that this concept has its sources in very different areas of scientific and medical thinking. Of great importance were:

(i)

a new theory of disease arising early in the 19th century supposing a continuous change from the healthy to the diseased state;

(ii)

the examination of variation within and between species of plants and animals;

(iii)

the clinical theory of constitution developed to describe the wholeness of the individual determined both by genetic factors and the influence of the environment;

(iv)

the development of mathematical and statistical tools starting with the adaptation of Bernoulli's “law of the great numbers” and Gauss' and Legendre's “law of errors” to biological measurements by the Belgian astronomer Quetelet.

At the end of the First World War the concept of normality was first discussed. An idealistic “value norm” was set against a statistical “frequency norm”. Between 1920 and 1930 the principles of our present concept were accepted and the mathematical tools developed. It took several decades to introduce this concept into practical medicine finally being recommended by the IFCC.
Blood and Tissue Findings in the Diagnosis of Mesenteric Ischaemia: An Experimental study
Authors: Aydın Metin , Güler Osman , Uǧras Serdar , Bakır Bahtiyar , Şekeroǧlu Ramazan
Published: October 2004

In this experimental study, after inducing mesenteric ischaemia in dogs, we investigated routine blood biochemistry, serum malondialdehyde (MDA) levels, histopathological changes and tissue MDA levels in the liver and intestines. The study group included 10 dogs. Six dogs which underwent sham operation served as controls. In the study group, the MDA level in liver tissue increased after superior mesenteric artery (SMA) ligation. Various degrees of necrosis were seen in the ileal biopsies 10 hours after the ligation. Hyperaemia and focal necrosis in the liver accompanied the intestinal necrosis if it was limited within the mucosa. However, massive or centrilobular necrosis was observed in the liver of those dogs which had intestinal necrosis that extended into muscular layers. MDA levels in intestinal tissue measured after SMA ligation were significantly higher than the preligation levels. On the other hand, the difference between serum and tissue MDA levels was not significant. The alterations in MDA were not significant in the sham group. Thus it is concluded that the serum MDA levels are valuable markers of diagnosis in intestinal ischaemia.
Erroneous Monocyte and Neutrophil Counts with the Sysmex NE-1500 in Patients Recovering from Bone Marrow Aplasia
Authors: Van Acker Jos , Wilmes Pascale , Philippé Jan
Published: October 2004

The Sysmex NE-1500 is a haematological analyser which offers five-part differential counts. The instrument was submitted to an extensive evaluation procedure before acceptance to the laboratory and was found to perform satisfactorily. However, problems arose in clinical practice with the analysis of samples from patients recovering from bone marrow aplasia. Falsely low monocyte counts and, controversely, falsely high neutrophil counts were found in these patients. Only a minority (13%) of these samples were flagged as abnormal by the instrument. The most alarming consequence of these results is the overestimation of neutrophils and the exclusion of these patients from reimbursement for treatment with growth factors. We conclude that in patients recovering from aplasia, differential counts performed with the Sysmex NE series should always be checked manually.
N-Benzoyl, L-Glutamic Acid as a Suitable Internal Standard for the Analysis of trans,trans-Muconic Acid in Human Urine by Liquid Chromatography
Authors: Renom Gilles , Bruneau Nathalie , Mizon Jacques
Published: October 2004

Urinary trans,trans-muconic acid is a sensitive biomarker for low level benzene exposure. The method described by Ducos et al. (Int Arch Occup Environ Health 1990; 62:529–34) is commonly used for its determination. In this study, we demonstrate that N-benzoyl, L-glutamic acid added to urine samples is a suitable internal standard to control trans,trans-muconic acid recovery after solid phase extraction of urine and to compensate for variations which might occur during high-performance liquid chromatography analysis.
Characterisation and Evaluation of External Quality Assessment Scheme Serum
Authors: Middle Jonathan G. , Libeer Jean-Claude , Malakhov Vladimir , Penttilä Ilkka
Published: October 2004

This work reviews the procurement, specification and manufacture of serum for external quality assessment (EQA) and provides a checklist for scheme organisers. Guidance on the assessment of commutability and notes on the use of EQA materials to assess assay problems are provided. The importance of the appropriateness of materials in respect of scheme design and objectives, and the essential nature of a detailed specification prior to manufacture are emphasised.
Comparison of Two Reverse Transcription-Polymerase Chain Reaction Methods for Detection of AML1/ETO Rearrangement in the M2 Subtype of Acute Myeloid Leukaemia
Authors: Barragán Eva , Bonanad Santiago , López José-Antonio , Bolufer Pascual , Sanz Miguel-Angel
Published: October 2004

Two reverse transcription-polymerase chain reaction methods to detect the AML1/ETO rearrangement in the M2 subtype of acute myeloid leukaemia those of Downing et al. (Blood 1993; 81:2860–5) and Satake et al.(Br J Haematol 1995; 91:892–8) were evaluated.

Bone marrow samples, one at diagnosis and two in complete remission from a patient with M2 subtype of acute myeloid leukaemia, with t(8;21), were analysed using both methods. The Kasumi-1 cell line was used as a positive control and a patient with M3 subtype of acute myeloid leukaemia as a negative control. To confirm the feasibility of Satake's method a group of 35 patients with subtypes of acute myeloid leukaemia at diagnosis were studied.

The method of Downing requires Southern blotting and hybridization with a specific probe because it often generates non-specific amplification products. By contrast, the method of Satake yields only a single amplification product, using one single round of PCR in samples at diagnosis, or two rounds in complete remission samples. The sensitivity of this method allows the detection of a single Kasumi-1 cell in 106 normal cells. The AML1/ETO rearrangement was observed in 5 of the 35 cases of acute myeloid leukaemia at diagnosis (14.3%) and in 3 of the 14 cases of M2 subtype of acute myeloid leukaemia (21.4%). The two remaining positive cases corresponded to the acute myeloid leukaemia subtypes M4 and M6. The results indicate that the method of Satake better meets the requirements of the clinical laboratory due to its greater simplicity, specificity, sensitivity and feasibility, thus making it more appropriate for use in diagnosing and monitoring minimal residual disease.
Oxidative Stress and Antioxidant Defenses in Renal Patients Receiving Regular Haemodialysis
Authors: Fiorillo Claudia , Oliviero Ciro , Rizzuti Giovanni , Nediani Chiara , Pacini Alessandra , Nassi Paolo
Published: October 2004

Patients with chronic renal failure, and particularly those receiving regular haemodialysis, have a high incidence of premature cardiovascular disease. Oxidative stress, which causes lipid peroxidation, may contribute to increase the risk of atherosclerosis. The results of the present study indicate that lipid peroxidation products (malonaldehyde and 4-hydroxyalkenals) are significantly increased in plasma of renal patients before dialysis and, although reduced, remained above the normal range after this treatment. Moreover, production of free radicals and reactive oxygen metabolites was increased in chronic renal failure patients, especially after dialysis. On the other hand, the antioxidant defenses of those patients were higher than those of normal subjects, as judged from the plasma levels of specific antioxidant molecules and from the plasma antioxidant capacity.

We also found that triglycerides were significantly higher in renal patients, both before and after dialysis, than in the control group.

These results suggest that patients on chronic haemodialysis are particularly prone to oxidative stress and that dialysis itself may worsen this condition. Rather than to a weakening of antioxidant defenses, the susceptibility of chronic renal failure patients to oxidative stress might be ascribed to an increased free radical and reactive oxygen metabolite production and to increased levels of oxidizable substrates, notably triglycerides with their unsaturated fatty acids.
Assessment of Serum Total and Bone Alkaline Phosphatase Measurement in Clinical Practice
Authors: Romagnoli Elisabetta , Minisola Giovanni , Carnevale Vincenzo , Scillitani Alfredo , Frusciante Vincenzo , Aliberti Giuseppe , Minisola Salvatore
Published: October 2004

The aim of the study was to measure serum levels of the bone-specific isoenzyme of alkaline phosphatase in normal subjects and patients with metabolic bone disease by using an immunoadsorption assay. We studied 140 healthy adults, 122 patients affected by metabolic bone disease and 15 patients with cholestatic liver disease.

Mean values of the bone-specific isoenzyme of alkaline phosphatase in healthy men were significantly higher than those found in premenopausal women (17.8 ± 4.2 U/l vs 15.6 ± 4.6 U/l, p<0.02); postmenopausal women had significantly higher levels of bone-specific isoenzyme of alkaline phosphatase (22.6 ± 6.4 U/l) than premenopausal women (p<0.0001). After the menopause total alkaline phosphatase increased by 46 %, while the increase in bone-specific isoenzyme of alkaline phosphatase was 39 %. No significant correlations were found between bone-specific isoenzyme of alkaline phosphatase and either age or years since menopause, in postmenopausal subjects. In patients with bone-specific isoenzyme of alkaline phosphatase above the upper limit of normal, the assay had a sensitivity of 100 % only in patients with Paget's disease of bone. In patients with cholestatic liver disease we found no correlation between bone-specific isoenzyme of alkaline phosphatase and either total alkaline phosphatase and γ-glutamyl transpeptidase, while a positive correlation was found between total alkaline phosphatase and γ-glutamyl transpeptidase.

Our results confirm the role of bone-specific isoenzyme of alkaline phosphatase assay in clinical research; however, its usefulness in clinical practice is unclear once liver involvement has been excluded.
Seeing and Photographing Your Visual Observations
Authors: Petermann MA
Published: October 2004

Light can play tricks on your eyes. Deficiencies that are observable at 1:00 PM are not necessarily observable at 3:00 PM. Depending on the nature of the light, sunlight vs. daylight, an inspector may or may not observe a bowing stone panel. Depending on the building facade material, configuration, or texture, light may or may not illuminate a facade deficiency. Ornate facades cast shadows in sunlight. A crack or spall can be lurking in those shadows. The inspector's choice of binoculars, experience, and possibly his psychological disposition may influence whether or not he observes an existing facade deficiency. Recognizing the deficiency may truly be a challenge, but photographing the deficiency may be a greater challenge. The crack or spall that lurks in the shadow on a sunny day may be nearly impossible to photograph. Excessive sunlight that reflects off the facade may wash out a photograph. Automatic cameras typically grab all surrounding light for a photograph, but this may be too much or too little depending upon the item to be photographed within the frame. The ability to set your camera manually may be necessary. This paper discusses the nature of light and suggestions for observing building facades and for recording deficiencies.
Microstructural Aspects of Irradiation Damage in A508 Gr 4N Forging Steel: Composition and Flux Effects
Authors: Burke MG , Stofanak RJ , Hyde JM , English CA , Server WL
Published: October 2004

Neutron irradiation can promote significant changes in the microstructure and associated mechanical properties of low alloy steels. In particular, irradiation can induce the formation of non-equilibrium phases and segregation, which may lead to a degradation in toughness. In this study, the microstructural changes caused by neutron irradiation have been characterized in A508 Grade (Gr) 4N-type steels (∼3.5% Ni) using a variety of state-of-the-art analytical techniques including 3D-Atom Probe Field-Ion Microscopy and Small Angle Neutron Scattering, along with post-irradiation annealing studies combining Positron Annihilation Lineshape Analysis and hardness measurements. Important differences between conventional and “superclean” A508 Gr 4N steel have been identified in this investigation. The data indicate that Ni is not the controlling factor in the irradiation damage behavior of these materials; rather, the Mn content of the steel is a dominant factor in the irradiation-induced microstructural development of solute-related hardening features.
Beneficial Reuse of Foundry Sands in Controlled Low Strength Material
Authors: Dingrando JS , Edil TB , Benson CH
Published: October 2004

A study was conducted to determine how the unconfined compressive strength and flow of flowable fills prepared with foundry sand depends on the bentonite content of the sand. The study showed that there are several advantages of using foundry sands with bentonite content > 6% as the fine aggregate in flowable fill. These advantages include: (i) lower long-term strength gain (making the design of excavatable mixtures simpler and less risky), (ii) less flow loss, (iii) fewer components and fewer interactions between components that are difficult to characterize, and (iv) a larger fraction of inexpensive foundry sand being used in the mixture. The unconfined compressive strength (UCS) of flowable fills prepared with foundry sands is sensitive to the water-cement ratio (W/C), at least when the W/C spans a broad range (4–11). Mixtures with W/C < 6.5 generally will have excessive UCS, whereas a suitable UCS is generally associated with W/C > 6.5. Bentonite content does not affect the UCS systematically, but it does have an indirect effect in that foundry sands with more bentonite require more water to flow, which affects strength. The amount of water required to achieve adequate flow primarily is a function of the bentonite content of the foundry sand. In general, as the bentonite content of the foundry sand increases, the water content of the mixture should increase correspondingly. The amount of fly ash has only a modest effect on the amount of water required. The most important factor affecting flow loss is the presence of cementitious fly ash in the mixture. Flow loss can be reduced appreciably by using a foundry sand with at least 6% bentonite so that fly ash fines need not be added to the mixture.
Properties of Controlled Low-Strength Materials Made with Wood Fly Ash
Authors: Naik TR , Kraus RN , Siddique R , Chun Y-M
Published: October 2004

Controlled low-strength materials (CLSM) were made in the field using one source of wood fly ash as a major component. CLSM Mixtures S-1 and S-3 contained cement, wood fly ash (81 and 89%, respectively, by mass of cementitious materials [Cm]), and sand; whereas, Mixture S-2 contained cement, wood fly ash (11% of Cm), Class C coal fly ash (67% of Cm), and sand. Mixtures S-1, S-2, and S-3 showed respective compressive strength values of 0.8, 0.3, and 0.6 MPa at 28 d, and 1.4, 14.4, and 1.0 MPa at one year. Combination of wood and coal fly ashes might have caused the drastic increase in the strength of Mixture S-2 at late ages. The respective water permeability values of Mixtures S-2 and S-3 decreased from 68 and 33 μm/s at 63 d to 6 and 12 μm/s at 227 d due to the improvement of microstructure of these CLSM mixtures.
Long Term Study of 23 Excavatable Tennessee Flowable Fill Mixtures
Authors: Crouch LK , Dotson VJ , Badoe DA , Maxwell RA , Dunn TR , Sparkman A
Published: October 2004

Twenty-three different EFF mixtures were placed in trenches simulating utility cuts during March through May of 2001. All EFF mixtures were tested for flow, unit weight, gravimetric air content, suitability for load application, and compressive strength development over time. The trenches were excavated in March 2003. Excavation difficulty was correlated with laboratory compressive strength for non-air-entrained mixtures. Bearing capacity estimates with the dynamic cone penetration apparatus were determined for all trenches. Nine EFF mixtures were used to assess the impact of Portland cement content and ASTM C 618 Class F fly ash content. Portland cement contents of 17.8, 26.7, and 35.6 kg/m3 and ASTM C 618 Class F fly ash contents of 178, 219.5, and 261 kg/m3 were used to evaluate the impact of component proportions. Proportions for the EFF mixtures were chosen using Kentucky Transportation Cabinet and Tennessee Ready Mixed Concrete Association (TRMCA) recommendations as well as a previous Tennessee Technological University research mixture. Six EFF mixtures were used to assess the impact of Portland cement content and high-unburned carbon fly ash content. Portland cement contents of 26.7 and 35.6 kg/m3 and high-unburned carbon fly ash contents of 219.5, 261, and 302.5 kg/m3 were used to evaluate the impact of component proportions. The influence of aggregate type on EFF mixtures was evaluated by using five different aggregate types in the EFF mixture recommended by TRMCA (26.7 kg/m3 Portland cement and 219.5 kg/m3 ASTM C 618 Class F fly ash). In addition, four comparison EFF mixtures were also used in the study (1 Tennessee Department of Transportation (TDOT) and 3 air-entrained EFF mixtures).
Field Demonstration Test on Construction and Strength of Flexible Pipe Drainage System Using Flowable Fill
Authors: Masada T , Sargand SM
Published: October 2004

A team at Ohio University recently completed a research project related to flowable fill. The main objective of the project was to evaluate the feasibility of constructing an economical drainage pipe system using a flexible thermoplastic pipe and flowable fill. The project tasks were divided into three phases (laboratory characterization tests, field demonstration tests, and engineering analysis). This technical paper summarizes mainly data obtained during one of the Phase 2 field demonstration tests conducted at the load frame site, utilizing a corrugated HDPE pipe, flowable fill, and a variety of sensors. The test results confirmed many previously cited advantages of using flowable fill as pipe backfill material and also showed that some potential problems could be overcome easily. Finite element analysis simulated the field performance of the flexible pipe-flowable fill system reasonably well. In summary, it was concluded that it was quite feasible to construct a sound subsurface drainage system using flexible pipe and flowable fill.
Skiboard Injuries—A Three-Year Comparison with Alpine Skiing
Authors: Langran M
Published: October 2004

Skiboarding is a relatively new snow sport that has increased in popularity in Scotland. All skiboard and alpine ski injuries over a three-year period at ski areas in Scotland were recorded and comparative data collected from uninjured skiboarders. 136 skiboard and 1362 alpine ski injuries were recorded, as well as data from 174 uninjured skiboarders. The profile of a typical skiboarder was a young male with little experience of the sport. Compared to alpine skiing, skiboarding was associated with a higher overall risk of injury and the highest fracture rate of any snow sport reported to date. Compared to alpine skiers, skiboarders sustained more injuries to the lower limb and higher rates of lower leg fractures. The nonrelease nature of a skiboard binding appears to be a major factor in the pattern of injuries seen on skiboards. Attention should be directed towards the development of an appropriate release binding mechanism for skiboards.
B-Myb and C-Myb Play Required Roles in Neuronal Apoptosis Evoked by Nerve Growth Factor Deprivation and DNA Damage
Authors: Liu David X. , Biswas Subhas C. , Greene Lloyd A.
Published: October 2004

Activation of cell cycle elements plays a required role in neuronal apoptosis associated with both development and neurodegenerative disorders. We demonstrated previously that neuron survival requires gene repression mediated by the cell cycle transcription factor E2F (E2 promoter binding factor) and that apoptotic stimuli lead to de-repression of E2F-regulated genes and consequent death. However, the downstream mediators of such death have been unclear. The transcription factorsB- andC-mybare E2F-regulated genes that are induced in neurons by apoptotic stimuli. Here, we examine the role of B- and C-myb induction in neuron death. Antisense and siRNA constructs that effectively block the upregulation of B- and C-myb provide substantial protection against death of cultured neuronal PC12 cells, sympathetic neurons, and cortical neurons elicited by either NGF withdrawal or DNA damage. There is also significant protection from death induced by direct E2F-dependent gene de-repression. Our findings thus establish required roles for B- and C-myb in neuronal apoptosis.
Molecular, Topographic, and Functional Organization of the Cerebellar Cortex: A Study with Combined Aldolase C and Olivocerebellar Labeling
Authors: Sugihara Izumi , Shinoda Yoshikazu
Published: October 2004

Aldolase C (zebrin) expression in Purkinje cells reveals stripe-shaped compartments in the cerebellar cortex. However, it is not clear how these compartments are related to cerebellar functional localization. Therefore, we identified olivocerebellar projections to aldolase C compartments by labeling climbing fibers with biotinylated dextran injected into various small areas within the inferior olive in rats. Specific rostral and caudal aldolase C compartments were linked in an orderly manner by common olivocerebellar projection across the rostrocaudal boundary on lobule VIc-crus Ib. Based on the localization of the olivary origins of projection to similar compartments, the compartments and olivocerebellar projections could be sorted into five groups: group I, positive compartments extending from the posterior lobe to the anterior lobe innervated by the principal olive and some neighboring areas; group II, positive compartments localized within the posterior lobe innervated by several medial subnuclei; group III, vermal and central negative compartments innervated by the centrocaudal medial accessory olive; group IV, negative and lightly positive compartments in the hemisphere and the rostral and caudal pars intermedia innervated by the dorsal accessory olive and some neighboring areas; group V, the flocculus and nodulus. The olivocerebellar topography within each group was simple and suggests an “orientation axis” within the concerned parts of the inferior olive. Furthermore, parts of the inferior olive in each group receive specific afferent inputs, indicating a close relationship between aldolase C compartments and functional localization. Thus, the five-group scheme we propose here may integrate the molecular, topographic, and functional organization of the cerebellum.
Neuropeptide Y Inhibits Hypocretin/Orexin Neurons by Multiple Presynaptic and Postsynaptic Mechanisms: Tonic Depression of the Hypothalamic Arousal System
Authors: Fu Li-Ying , Acuna-Goycolea Claudio , van den Pol Anthony N.
Published: October 2004

Neurons that release neuropeptide Y (NPY) have important effects on hypothalamic homeostatic regulation, including energy homeostasis, and innervate hypocretin neurons. Using whole-cell patch-clamp recording, we explored NPY actions on hypocretin cells identified by selective green fluorescent protein expression in mouse hypothalamic slices. NPY reduced spike frequency and hyperpolarized the membrane potential of hypocretin neurons. The NPY hyperpolarizing action persisted in tetrodotoxin (TTX), was mimicked by Y1 receptor-selective agonists [Pro34]-NPY and [d-Arg25]-NPY, and was abolished by the Y1-specific antagonist BIBP3226 [(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-d-arginine-amide], consistent with a direct activation of postsynaptic Y1 receptors. NPY induced a current that was dependent on extracellular potassium, reversed near the potassium equilibrium potential, showed inward rectification, was blocked by extracellular barium, and was abolished by GDP-βS in the recording pipette, consistent with a G-protein-activated inwardly rectifying K+(GIRK) current. [Pro34]-NPY evoked, and BIBP3226 blocked, the activation of the GIRK-type current, indicating mediation by a Y1 receptor. NPY attenuated voltage-dependent calcium currents mainly via a Y1 receptor subtype. BIBP3226 increased spontaneous spike frequency, suggesting an ongoing Y1 receptor-mediated NPY inhibition. In TTX, miniature EPSCs were reduced in frequency but not amplitude by NPY, NPY13-36, and [d-Trp32]-NPY, but not by [Pro34]-NPY, suggesting the presynaptic inhibition was mediated by a Y2/Y5 receptor. NPY had little effect on GABA-mediated miniature IPSCs but depressed spontaneous IPSCs. Together, these data support the view that NPY reduces the activity of hypocretin neurons by multiple presynaptic and postsynaptic mechanisms and suggest NPY axons innervating hypocretin neurons may tonically attenuate hypocretin-regulated arousal.
Regulation of Neuronal Excitability through Pumilio-Dependent Control of a Sodium Channel Gene
Authors: Mee Christopher J. , Pym Edward C. G. , Moffat Kevin G. , Baines Richard A.
Published: October 2004

Dynamic changes in synaptic connectivity and strength, which occur during both embryonic development and learning, have the tendency to destabilize neural circuits. To overcome this, neurons have developed a diversity of homeostatic mechanisms to maintain firing within physiologically defined limits. In this study, we show that activity-dependent control of mRNA for a specific voltage-gated Na+channel [encoded byparalytic(para)] contributes to the regulation of membrane excitability inDrosophilamotoneurons. Quantification ofparamRNA, by real-time reverse-transcription PCR, shows that levels are significantly decreased in CNSs in which synaptic excitation is elevated, whereas, conversely, they are significantly increased when synaptic vesicle release is blocked. Quantification of mRNA encoding the translational repressorpumilio(pum) reveals a reciprocal regulation to that seen forpara. Pumilio is sufficient to influenceparamRNA. Thus,paramRNA is significantly elevated in a loss-of-function allele ofpum(pumbemused), whereas expression of a full-lengthpumtransgene is sufficient to reduceparamRNA. In the absence ofpum, increased synaptic excitation fails to reduceparamRNA, showing that Pum is also necessary for activity-dependent regulation ofparamRNA. Analysis of voltage-gated Na+current (INa) mediated byparain two identified motoneurons (termed aCC and RP2) reveals that removal ofpumis sufficient to increase one of two separableINacomponents (persistentINa), whereas overexpression of apumtransgene is sufficient to suppress both components (transient and persistent). We show, through use of anemone toxin (ATX II), that alteration in persistentINais sufficient to regulate membrane excitability in these two motoneurons.
Histamine H3Receptors Inhibit Serotonin Release in Substantia Nigra Pars Reticulata
Authors: Threlfell Sarah , Cragg Stephanie J. , Kalló Imre , Turi Gergely F. , Coen Clive W. , Greenfield Susan A.
Published: October 2004

The substantia nigra pars reticulata (SNr) plays a key role in basal ganglia function. Projections from multiple basal ganglia nuclei converge at the SNr to regulate nigrothalamic output. The SNr is also characterized by abundant aminergic input, including dopaminergic dendrites and axons containing 5-hydroxytryptamine (5-HT) or histamine (HA). The functions of HA in the SNr include motor control via HA H3receptors (H3Rs), although the mechanism remains far from elucidated. In Parkinson's disease, there is an increase in H3Rs and the density of HA-immunoreactive axons in the SN.

We explored the role of H3Rs in the regulation of 5-HT release in SNr using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in rat midbrain slices. Immunohistochemistry identified a similar distribution for histaminergic and serotonergic processes in the SNr: immunoreactive varicosities were observed in the vicinity of dopaminergic dendrites. Electrically evoked 5-HT release was dependent on extracellular Ca2+and prevented by NaV+-channel blockade. Extracellular 5-HT concentration was enhanced by inhibition of uptake transporters for 5-HT but not dopamine. Selective H3R agonists (R)-(-)-α-methyl-histamine or immepip inhibited evoked 5-HT release by up to 60%. This inhibition was prevented by the H3R antagonist thioperamide but not by the 5-HT1Breceptor antagonist isamoltane. H3R inhibition of 5-HT release prevailed in the presence of GABA or glutamate receptor antagonists (ionotropic and metabotropic), suggesting minimal involvement of GABA or glutamate synapses. The potent regulation of 5-HT by H3Rs reported here not only elucidates HA function in the SNr but also raises the possibility of novel targets for basal ganglia therapies.
Switching Mature Retinal Ganglion Cells to a Robust Growth StateIn Vivo: Gene Expression and Synergy with RhoA Inactivation
Authors: Fischer Dietmar , Petkova Victoria , Thanos Solon , Benowitz Larry I.
Published: October 2004

The inability of mature CNS neurons to regenerate injured axons has been attributed to a loss of inherent growth potential of cells and to inhibitory signals associated with myelin and the glial scar. The present study investigated two complementary issues: (1) whether mature CNS neurons can be stimulated to alter their gene expression profile and switch into a strong growth state; and (2) whether inactivating RhoA, a convergence point for multiple inhibitory signals, is sufficient to produce strong regeneration even without activating the growth state of neurons. In the mature rat, retinal ganglion cells (RGCs) normally fail to regenerate axons through the injured optic nerve but can be stimulated to do so by activating macrophages in the eye (e.g., by lens injury). To investigate underlying changes in gene expression, we retrogradely labeled RGCs with a fluorescent dye, performed optic nerve surgery with or without lens injury, and 4 d later, dissociated retinas, isolated RGCs by fluorescence-activated cell sorting, and examined their profiles of gene expression using microarrays. To investigate the effects of inactivating RhoA, we transfected RGCs with adeno-associated viruses carrying a gene for C3 ribosyltransferase. Our results show that, with appropriate stimulation, mature CNS neurons can undergo dramatic changes in gene expression comparable with those seen in regenerating neurons of the PNS, and that RhoA inactivation by itself results in moderate regeneration, and strongly potentiates axon regeneration through the mature optic nerve when the growth state of neurons is activated.
A New Platform to Study the Molecular Mechanisms of Exocytosis
Authors: Mezer Aviv , Nachliel Esther , Gutman Menachem , Ashery Uri
Published: October 2004

The exocytotic process in neurons and neuroendocrine cells consists of a sequence of reactions between well defined proteins. In the present study, we have created for the first time a comprehensive kinetic model that demonstrates the dynamics of interactions between key synaptic proteins that are associated with exocytosis. The interactions between the synaptic proteins were transformed into differential rate equations that, after their integration over time, reconstructed the experimental signal. The model can perfectly reconstruct the kinetics of exocytosis, the calcium-dependent priming and fusion processes, and the effects of genetic manipulation of synaptic proteins. The model suggests that fusion occurs from two parallel pathways and assigns precise, non-identical synaptic protein complexes to the two pathways. In addition, it provides a unique opportunity to study the dynamics of intermediate protein complexes during the fusion process, a possibility that is hidden in most experimental systems. We thus developed a novel approach that allows detailed characterization of the temporal relationship between synaptic protein complexes. This model provides an excellent platform for prediction and quantification of the effects of protein manipulations on exocytosis and opens new avenues for experimental investigation of exocytosis.
The ZebrafishnrcMutant Reveals a Role for the Polyphosphoinositide Phosphatase Synaptojanin 1 in Cone Photoreceptor Ribbon Anchoring
Authors: Van Epps Heather A. , Hayashi Mitsuko , Lucast Louise , Stearns George W. , Hurley James B. , De Camilli Pietro , Brockerhoff Susan E.
Published: October 2004

Visual, vestibular, and auditory neurons rely on ribbon synapses for rapid continuous release and recycling of synaptic vesicles. Molecular mechanisms responsible for the properties of ribbon synapses are mostly unknown. The zebrafish vision mutantnrchas unanchored ribbons and abnormal synaptic transmission at cone photoreceptor synapses. We used positional cloning to identify thenrcmutation as a premature stop codon in thesynaptojanin1(synj1) gene. Synaptojanin 1 (Synj1) is undetectable innrcextracts, and biochemical activities associated with it are reduced. Furthermore, morpholinos directed againstsynj1phenocopy thenrcmutation. Synj1 is a polyphosphoinositide phosphatase important at conventional synapses for clathrin-mediated endocytosis and actin cytoskeletal rearrangement. In thenrccone photoreceptor pedicle, not only are ribbons unanchored, but synaptic vesicles are reduced in number, abnormally distributed, and interspersed within a dense cytoskeletal matrix. Our findings reveal a new role for Synj1 and link phosphoinositide metabolism to ribbon architecture and function at the cone photoreceptor synapse.
Calcium-Activated Potassium Channels Are Selectively Coupled to P/Q-Type Calcium Channels in Cerebellar Purkinje Neurons
Authors: Womack Mary D. , Chevez Carolyn , Khodakhah Kamran
Published: October 2004

Cerebellar Purkinje neurons fire spontaneously in the absence of synaptic transmission. P/Q-type voltage-gated calcium channels and calcium-activated potassium channels are required for normal spontaneous activity. Blocking P/Q-type calcium channels paradoxically mimics the effects of blocking calcium-activated potassium channels. Thus, an important function of the P/Q-type calcium channels is to provide calcium for activation of calcium-activated potassium channels. Purkinje neurons express several classes of voltage-gated calcium channels, and the P/Q- and T-type channels make comparable contributions to total calcium entry after an action potential. Here we demonstrate that calcium-activated potassium channels are activated exclusively by calcium entering through P/Q-type voltage-gated calcium channels. This selective coupling is maintained even when calcium flux through voltage-gated channels is increased by increasing the extracellular calcium concentration. Small decreases in P/Q current density are likely to alter spontaneous activity of Purkinje neurons via decreased recruitment of calcium-activated potassium channels. In both human and murine animal models, mutations that decrease P/Q current density in Purkinje neurons also cause cerebellar ataxia. Alterations in the spontaneous activity of Purkinje neurons may be an important contributing factor to the ataxia in these subjects.
Robustness of Multiplicative Processes in Auditory Spatial Tuning
Authors: Peña José Luis , Konishi Masakazu
Published: October 2004

Auditory space-specific neurons in the owl's inferior colliculus selectively respond to the direction of sound propagation, which is defined by combinations of interaural time (ITD) and level (ILD) differences. Mathematical analyses show that the amplitude of postsynaptic potentials in these neurons is a product of two components that vary with either ITD or ILD. Temporal correlation in the fine structure of signals between the ears is essential for detection of ITD. By varying the degree of binaural correlation, we could accurately change the amplitude of the ITD component of postsynaptic potentials in the space-specific neurons. Multiplication worked for the entire range of postsynaptic potentials created by manipulation of ITD.
Ral and Phospholipase D2-Dependent Pathway for Constitutive Metabotropic Glutamate Receptor Endocytosis
Authors: Bhattacharya Moshmi , Babwah Andy V. , Godin Christina , Anborgh Pieter H. , Dale Lianne B. , Poulter Michael O. , Ferguson Stephen S. G.
Published: October 2004

G-protein-coupled receptors play a central role in the regulation of neuronal cell communication. Class 1 metabotropic glutamate receptors (mGluRs) mGluR1a and mGluR5a, which are coupled with the hydrolysis of phosphoinositides, are essential for modulating excitatory neurotransmission at glutamatergic synapses. These receptors are constitutively internalized in heterologous cell cultures, neuronal cultures, and intact neuronal tissues. We show here that the small GTP-binding protein Ral, its guanine nucleotide exchange factor RalGDS (Ral GDP dissociation stimulator), and phospholipase D2 (PLD2) are constitutively associated with class 1 mGluRs and regulate constitutive mGluR endocytosis. Moreover, both Ral and PLD2 are colocalized with mGluRs in endocytic vesicles in both human embryonic kidney 293 (HEK 293) cells and neurons. Ral and PLD2 activity is required for the internalization of class 1 mGluRs but is not required for the internalization of the β2-adrenergic receptor. Constitutive class 1 mGluR internalization is not dependent on the downstream Ral effector proteins Ral-binding protein 1 and PLD1 or either ADP-ribosylation factors ARF1 or ARF6. The treatment of HEK 293 cells and neurons with small interfering RNA both downregulates PLD2 expression and blocks mGluR1a and mGluR5a endocytosis. The constitutive internalization of mGluR1a and mGluR5a is also attenuated by the treatment of cells with 1-butanol to prevent PLD2-mediated phosphatidic acid formation. We propose that the formation of a mGluR-scaffolded RalGDS/Ral/PLD2 protein complex provides a novel alternative mechanism to β-arrestins for the constitutive endocytosis of class 1 mGluRs.