Differential expression and modification of neurofilament triplet proteins during cat cerebellar development.

Neurofilament (NF) proteins consist of three subunits of different molecular weights defined as NF-H, NF-M, and NF-L. They are typical structures of the neuronal cytoskeleton. Their immunocytochemical distribution during postnatal development of cat cerebellum was studied with several monoclonal and polyclonal antibodies against phosphorylated or unmodified sites. Expression and distribution of the triplet neurofilament proteins changed with maturation. Afferent mossy and climbing fibers in the medullary layer contained NF-M and NF-L already at birth, whereas NF-H appeared later. Within the first three postnatal weeks, all three subunits appeared in mossy and climbing fibers in the internal granular and molecular layers and in the axons of Purkinje cells. Axons of local circuit neurons such as basket cells expressed these proteins at the end of the first month, whereas parallel fibers expressed them last, at the beginning of the third postnatal month. Differential localization was especially observed for NF-H. Depending on phosphorylation, NF-H proteins were found in different axon types in climbing, mossy, and basket fibers or additionally in parallel fibers. A nonphosphorylated NF-H subunit was exclusively located in some Purkinje cells at early developmental stages and in some smaller interneurons later. A novel finding is the presence of a phosphorylation site in the NF-H subunit that is localized in dendrites of Purkinje cells but not in axons. Expression and phosphorylation of the NF-H subunit, especially, is cell-type specific and possibly involved in the adult-type stabilization of the axonal and dendritic cytoskeleton.

Neurofilament triplet proteins are restricted to a subset of neurons in the rat neocortex.

The cellular localisation of neurofilament triplet subunits was investigated in the rat neocortex. A subset of mainly pyramidal neurons showed colocalisation of subunit immunolabelling throughout the neocortex, including labelling with the antibody SMI32, which has been used extensively in other studies of the primate cortex as a selective cellular marker. Neurofilament-labelled neurons were principally localised to two or three cell layers in most cortical regions, but dramatically reduced labelling was present in areas such as the perirhinal cortex, anterior cingulate and a strip of cortex extending from caudal motor regions through the medial parietal region to secondary visual areas. However, quantitative analysis demonstrated a similar proportion (10-20%) of cells with neurofilament triplet labelling in regions of high or low labelling. Combining retrograde tracing with immunolabelling showed that cellular content of the neurofilament proteins was not correlated with the length of projection. Double labelling immunohistochemistry demonstrated that neurofilament content in axons was closely associated with myelination. Analysis of SMI32 labelling in development indicated that content of this epitope within cell bodies was associated with relatively late maturation, between postnatal days 14 and 21. This study is further evidence of a cell type-specific regulation of neurofilament proteins within neocortical neurons. Neurofilament triplet content may be more closely related to the degree of myelination, rather than the absolute length, of the projecting axon.

Regulation of recombination at yeast nuclear pores controls repair and triplet repeat stability.

Secondary structure-forming DNA sequences such as CAG repeats interfere with replication and repair, provoking fork stalling, chromosome fragility, and recombination. In budding yeast, we found that expanded CAG repeats are more likely than unexpanded repeats to localize to the nuclear periphery. This positioning is transient, occurs in late S phase, requires replication, and is associated with decreased subnuclear mobility of the locus. In contrast to persistent double-stranded breaks, expanded CAG repeats at the nuclear envelope associate with pores but not with the inner nuclear membrane protein Mps3. Relocation requires Nup84 and the Slx5/8 SUMO-dependent ubiquitin ligase but not Rad51, Mec1, or Tel1. Importantly, the presence of the Nup84 pore subcomplex and Slx5/8 suppresses CAG repeat fragility and instability. Repeat instability in nup84, slx5, or slx8 mutant cells arises through aberrant homologous recombination and is distinct from instability arising from the loss of ligase 4-dependent end-joining. Genetic and physical analysis of Rad52 sumoylation and binding at the CAG tract suggests that Slx5/8 targets sumoylated Rad52 for degradation at the pore to facilitate recovery from acute replication stress by promoting replication fork restart. We thereby confirmed that the relocation of damage to nuclear pores plays an important role in a naturally occurring repair process.

Right-left digit ratio (2D:4D) and maximal oxygen uptake.

A low digit ratio (2D:4D) and low 2D:4D in the right compared with the left hand (right-left 2D:4D) are thought to be determined by high in utero concentrations of testosterone, and are related to "masculine" traits such as aggression and performance in sports like running and rugby. Low right-left 2D:4D is also related to sensitivity to testosterone as measured by the number of cytosine-adenine-guanine triplet repeats in exon 1 of the androgen receptor gene. Here we show that low right-left 2D:4D is associated with high maximal oxygen uptake (VO2(max)), high velocity at VO2(max), and high maximum lactate concentration in a sample of teenage boys. We suggest that low right-left 2D:4D is linked to performance in some sports because it is a proxy of high sensitivity to prenatal and maybe also circulating testosterone and high VO2(max).

A novel neuro-muscular marker for the heavy subunit of neurofilament proteins, NF-H, and striated muscle myosin of Xenopus laevis.

A novel monoclonal antibody, M7, is described, that reacts on Western blots with the large subunit of the neurofilament triplet proteins (NF-H) and with striated muscle myosin of Xenopus laevis. Enzymatically digested neurofilament and myosin proteins revealed different immunoreactive peptide fragments on Western blots. Therefore, the antibody must react with immunologically related epitopes common to both proteins. Immunohistochemistry showed staining of large and small axons in CNS and PNS, and nerves could be followed into endplate regions of skeletal muscles. These muscles were characterized by a striated immunostaining of the M-lines. Despite the crossreactivity of M7 with NF-H and muscle myosin, this antibody may be a tool to study innervation of muscle fibers, and to define changes in the neuromuscular organization during early development and metamorphosis of tadpoles.

Individual monitoring of internal exposure for nuclear medicine workers in Switzerland.

Monitoring of internal exposure for nuclear medicine workers requires frequent measurements due to the short physical half-lives of most radionuclides used in this field. The aim of this study was to develop screening measurements performed at the workplace by local staff using standard laboratory instrumentation, to detect whether potential intake has occurred. Such measurements do not enable to determine the committed effective dose, but are adequate to verify that a given threshold is not exceeded. For radioiodine, i.e. (123)I, (124)I, (125)I and (131)I, a calibrated surface contamination monitor is placed in front of the thyroid to detect whether the activity threshold has been exceeded. For radionuclides with very short physical half-lives (≤6 h), such as (99m)Tc and those used in positron emission tomography  imaging, i.e. (11)C, (15)O, (18)F and (68)Ga, screening procedures consist in performing daily measurements of the ambient dose rate in front of the abdomen. Other gamma emitters used for imaging, i.e. (67)Ga, (111)In and (201)Tl, are measured with a scintillation detector located in front of the thorax. For pure beta emitters, i.e. (90)Y and (169)Er, as well as beta emitters with low-intensity gamma rays, i.e. (153)Sm, (177)Lu, (186)Re and (188)Re, the procedure consists in measuring hand contamination immediately after use. In Switzerland, screening procedures have been adopted by most nuclear medicine services since such measurements enable an acceptable monitoring while taking into account practical and economic considerations.

Selective changes in the neurofilament and microtubule cytoskeleton of NF-H/LacZ mice.

This study focused mainly on changes in the microtubule cytoskeleton in a transgenic mouse where beta-galactosidase fused to a truncated neurofilament subunit led to a decrease in neurofilament triplet protein expression and a loss in neurofilament assembly and abolished transport into neuronal processes in spinal cord and brain. Although all neurofilament subunits accumulated in neuronal cell bodies, our data suggest an increased solubility of all three subunits, rather than increased precipitation, and point to a perturbed filament assembly. In addition, reduced neurofilament phosphorylation may favor an increased filament degradation. The function of microtubules seemed largely unaffected, in that tubulin and microtubule-associated proteins (MAP) expression and their distribution were largely unchanged in transgenic animals. MAP1A was the only MAP with a reduced signal in spinal cord tissue, and differences in immunostaining in various brain regions corroborate a relationship between MAP1A and neurofilaments.

Pi*-pi* bonding interactions generated by halogen oxidation of zirconium(IV) redox-active ligand complexes.

The new complex, [Zr(pda)2]n (1, pda2- = N,N'-bis(neo-pentyl)-ortho-phenylenediamide, n = 1 or 2), prepared by the reaction of 2 equiv of pdaLi2 with ZrCl4, reacts rapidly with halogen oxidants to afford the new product ZrX2(disq)2 (3, X = Cl, Br, I; disq- = N,N'-bis(neo-pentyl)-ortho-diiminosemiquinonate) in which each redox-active ligand has been oxidized by one electron. The oxidation products 3a-c have been structurally characterized and display an unusual parallel stacked arrangement of the disq- ligands in the solid state, with a separation of approximately 3 A. Density functional calculations show a bonding-type interaction between the SOMOs of the disq- ligands to form a unique HOMO while the antibonding linear combination forms a unique LUMO. This orbital configuration leads to a closed-shell-singlet ground-state electron configuration (S = 0). Temperature-dependent magnetism measurements indicate a low-lying triplet excited state at approximately 750 cm-1. In solution, 3a-c show strong disq--based absorption bands that are invariant across the halide series. Taken together these spectroscopic measurements provide experimental values for the one- and two-electron energies that characterize the pi-stacked bonding interaction between the two disq- ligands.

Differential vulnerability of neurochemically identified subpopulations of retinal neurons in a monkey model of glaucoma.

The vulnerability of subpopulations of retinal neurons delineated by their content of cytoskeletal or calcium-binding proteins was evaluated in the retinas of cynomolgus monkeys in which glaucoma was produced with an argon laser. We quantitatively compared the number of neurons containing either neurofilament (NF) protein, parvalbumin, calbindin or calretinin immunoreactivity in central and peripheral portions of the nasal and temporal quadrants of the retina from glaucomatous and fellow non-glaucomatous eyes. There was no significant difference between the proportion of amacrine, horizontal and bipolar cells labeled with antibodies to the calcium-binding proteins comparing the two eyes. NF triplet immunoreactivity was present in a subpopulation of retinal ganglion cells, many of which, but not all, likely correspond to large ganglion cells that subserve the magnocellular visual pathway. Loss of NF protein-containing retinal ganglion cells was widespread throughout the central (59-77% loss) and peripheral (96-97%) nasal and temporal quadrants and was associated with the loss of NF-immunoreactive optic nerve fibers in the glaucomatous eyes. Comparison of counts of NF-immunoreactive neurons with total cell loss evaluated by Nissl staining indicated that NF protein-immunoreactive cells represent a large proportion of the cells that degenerate in the glaucomatous eyes, particularly in the peripheral regions of the retina. Such data may be useful in determining the cellular basis for sensitivity to this pathologic process and may also be helpful in the design of diagnostic tests that may be sensitive to the loss of the subset of NF-immunoreactive ganglion cells.

Frequency-dependent attenuation as a potential indicator of oil saturation

At seismic frequencies, wave-induced fluid flow is a major cause of P-wave attenuation in partially saturated porous rocks. Attenuation is of great importance for the oil industry in the interpretation of seismic field data. Here, the effects on P-wave attenuation resulting from changes in oil saturation are studied for media with coexisting water, oil, and gas. For that, creep experiments are numerically simulated by solving Biot's equations for consolidation of poroelastic media with the finite-element method. The experiments yield time-dependent stress?strain relations that are used to calculate the complex P-wave modulus from which frequency-dependent P-wave attenuation is determined. The models are layered media with periodically alternating triplets of layers. Models consisting of triplets of layers having randomly varying layer thicknesses are also considered. The layers in each triplet are fully saturated with water, oil, and gas. The layer saturated with water has lower porosity and permeability than the layers saturated with oil and gas. These models represent hydrocarbon reservoirs in which water is the wetting fluid preferentially saturating regions of lower porosity. The results from the numerical experiments showed that increasing oil saturation, connected to a decrease in gas saturation, resulted in a significant increase of attenuation at low frequencies (lower than 2 Hz). Furthermore, replacing the oil with water resulted in a distinguishable behavior of the frequency-dependent attenuation. These results imply that, according to the physical mechanism of wave-induced fluid flow, frequency-dependent attenuation in media saturated with water, oil, and gas is a potential indicator of oil saturation.

A new method for the determination of plutonium and americium using high pressure microwave digestion and alpha-spectrometry or ICP-SMS

Plutonium and americium are radionuclides particularly difficult to measure in environmental samples because they are alpha-emitters and therefore necessitate a careful separation before any measurement, either using radiometric methods or ICP-SMS. Recent developments in extraction chromatography resins such as Eichrom (R) TRU and TEVA have resolved many of the analytical problems but drawbacks such as low recovery and spectral interferences still occasionally occur. Here, we report on the use of the new Eichrom (R) DGA resin in association with TEVA resin and high pressure microwave acid leaching for the sequential determination of plutonium and americium in environmental samples. The method results in average recoveries of 83 +/- 15% for plutonium and 73 +/- 22% for americium (n = 60), and a less than 10% deviation from reference values of four IAEA reference materials and three samples from intercomparisons exercises. The method is also suitable for measuring Pu-239 in water samples at the mu Bq/l level, if ICP-SMS is used for the measurement.

Fluorodeoxyuridine mediated cell cycle synchronization in S-phase increases the Auger radiation cell killing with 125I-iododeoxyuridine.

Aim: 125I-iododeoxyuridine is a potential Auger radiation therapy agent. Its incorporation in DNA of proliferating cells is enhanced by fluorodeoxyuridine. Here, we evaluated therapeutic activities of 125I-iododeoxyuridine in an optimized fluorodeoxyuridine pre-treatment inducing S-phase synchronization. Methods: After S-phase synchronization by fluorodeoxyuridine, cells were treated with 125I-iododeoxyuridine. Apoptosis analysis and S-phase synchronization were studied by flow cytometry. Cell survival was determined by colony-forming assay. Based on measured growth parameters, the number of decays per cell that induced killing was extrapolated. Results: Treatment experiments showed that 72 to 91% of synchronized cells were killed after 0.8 and 8 kBq/ml 125I-iododeoxyuridine incubation, respectively. In controls, only 8 to 38% of cells were killed by corresponding 125I-iododeoxyuridine activities alone and even increasing the activity to 80 kBq/ml gave only 42 % killing. Duplicated treatment cycles or repeated fluorodeoxyuridine pre-treatment allowed enhancing cell killing to >95 % at 8 kBq/ml 125I-iododeoxyuridine. About 50 and 160 decays per S-phase cells in controls and S-phase synchronization, respectively, were responsible for the observed cell killing at 0.8 kBq/ml radio-iododeoxyuridine. Conclusion: These data show the successful application of fluorodeoxyuridine that provided increased 125I-iododeoxyuridine Auger radiation cell killing efficacy through S-phase synchronization and high DNA incorporation of radio-iododeoxyuridine.

Moeglichkeiten der systemischen Therapie im metastasierten Stadium [Treatment options for chemotherapy in metastatic gastric cancer]

Die Ergebnisse mehrerer, in letzter Zeit publizierter Phase-III-Studien haben die therapeutischen Möglichkeiten in der Behandlung des metastasierten Magenkarzinoms deutlich erweitert. Die Dauerinfusion von 5-Fluorouracil (5-FU) kann ohne Verlust an Wirkung durch Capecitabin ersetzt werden, ebenso wie Cisplatin durch Oxaliplatin. Nach den Ergebnissen der REAL-2-Studie zeigt die Kombination aus Epirubicin, Oxaliplatin und Capecitabin (EOX) eine Verbesserung des Gesamtüberlebens (9,9 vs. 11,2 Monate; HR 0,8) im Vergleich zu Epirubicin, Cisplatin und 5-FU (ECF). Die Frage, ob in der First-Line-Therapie eine Dreifachkombination oder eine Zweifachkombination eingesetzt werden sollte, ist allerdings weiterhin umstritten. Die Kombination aus Irinotecan und 5-FU stellt für solche Patienten, bei denen aufgrund von Komorbiditäten eine platinfreie Therapie bevorzugt wird, eine Alternative zur Kombination Cisplatin/5-FU dar. Docetaxel, 5-FU und Cisplatin (DCF) hat sich bezüglich des Überlebens in einer randomisierten Phase-III-Studie als statistisch signifikant überlegen erwiesen, allerdings besteht eine ausgeprägte hämatologische Toxizität, welche die Anwendbarkeit insbesondere bei den häufig älteren Patienten mit einem Magenkarzinom limitiert. Randomisierte Phase-III-Studien zum Vergleich von DCF mit anderen Dreierkombinationen, wie z. B. EOX, stehen aus. Recently published results from several phase III trials have significantly increased the therapeutic options in the treatment of metastatic stomach cancer: The continuous infusion of 5-FU can be replaced by capecitabine, and cisplatin can be replaced by oxaliplatin in both cases without impairing efficacy. According to the results of the REAL-2 trial, the combination of epirubicin, oxaliplatin and capecitabine (EOX) achieved superior results for overall survival compared to epirubicin, cisplatin und 5-FU (ECF) (9.9 versus 11.2 months, HR 0.8). However, the question of whether an optimal first line therapy should include a triplet regimen or the sequential use of doublets is a matter of debate. The combination of irinotecan and 5-FU may serve as an alternative to platinum-containing regimens in patients where, due to co-morbidity, a platinum-free regimen is preferred. The 3-drug combination of docetaxel, 5-FU and cisplatin (DCF) demonstrated a statistically significant survival benefit compared to the 2-drug combination of 5-FU and cisplatin in a randomized phase III trial, although results were limited by a particularly significant hematological toxicity, which prevents its application in the large group of elderly patients with gastric cancer. Direct randomized phase III comparisons of DCF with other 3-drug combinations, such as EOX are still missing.

Delayed delivery of second twin: a multicentre study of 35 cases.

OBJECTIVE: The aim of this study was to conduct a statistical analysis to determine the outcome of conservative treatment after delivery of a first fetus in multiple pregnancy and thus define new prognostic factors. STUDY DESIGN: Multicentre retrospective study involving 12 centers over a 10-year period. RESULTS: Twenty-eight twin pregnancies and seven triplet pregnancies which were managed conservatively. In twin pregnancies, 79% of the delayed-delivery fetuses survived; only 7% of the first delivered fetuses survived. The mean interval between deliveries was 47 days. No statistical difference was found concerning cerclage, antibiotic therapy, tocolysis and hospitalization. Earlier delivery of the first twin and premature rupture of membranes for the second twin were significantly related to a longer interval between deliveries. CONCLUSION: Delayed delivery in multifetal pregnancies can be successful if there are no contraindications and these pregnancies are managed in a tertiary perinatal center. Publications limited to successful cases have undoubtedly introduced some bias in assessment.

Intraperikaryal neurofilamentous accumulations in a subset of retinal ganglion cells in aged mice that express a human neurofilament gene.

Neurofilamentous changes in select groups of neurons are associated with the degenerative changes of many human age-related neurodegenerative diseases. To examine the possible effects of aging on the neuronal cytoskeleton containing human proteins, the retinas of transgenic mice expressing the gene for the human middle-sized neurofilament triplet were investigated at 3 or 12 months of age. Transgenic mice developed tangle-like neurofilamentous accumulations in a subset of retinal ganglion cells at 12 months of age. These neurofilamentous accumulations, which also involved endogenous neurofilament proteins, were present in the perikarya and proximal processes of large ganglion cells and were predominantly located in peripheral retina. The presence of the human protein may thus confer vulnerability of the cytoskeleton to age-related alterations in this specific retinal cell type and may serve as a model for similar cellular changes associated with Alzheimer's disease and glaucoma.