bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlated analysis of cellular DNA, membrane antigens and light scatter of human lymphoid cells
Authors: Braylan Raul C. , Benson Neal A. , Nourse Virginia , Kruth Howard S.
Published: June 2010

Flow cytometric correlated analysis of membrane antigens, DNA, and light scatter was performed on human lymphoid cells using fluorescein (FITC)‐conjugated antibodies to label B‐ and T‐cell antigens and propidium iodide (PI) to stain DNA after ethanol fixation and RNase treatment. A FACS II flow cytometer was modified to obtain digitized measurements of two color fluorescence and light scatter emissions, simultaneously. Software was written to allow single parameter analysis or correlated analysis of any two of the three parameters acquired. Ethanol fixation preserved FITC surface labeling for at least 15 weeks, but produced marked changes in light scatter. No changes in FITC distributions were observed after RNase treatment and PI staining, and the presence of FITC labeling did not affect DNA distributions. Within heterogeneous cell populations, the DNA distribution of cell subpopulations identified by a membrane antigen was clearly demonstrated.
Caution on the use of lymphocytes as standards in the flow cytometric analysis of cultured cells
Authors: Wolley Robert C. , Herz Fritz , Koss Leopold G.
Published: June 2010

Flow cytometry was used to compare the fluorescence intensity of the G0/G1 peak of a diploid (WI‐38), a hypodiploid (C4I) and a heteroploid (KB) human cell line with that of nonstimulated human lymphocytes. With all three cultured cells the fluorescence intensity was substantially higher than that of lymphocytes. These results, obtained with three different DNA‐intercalating fluorochromes, suggest that lymphocytes may not be the standard of choice for flow cytometric studies of the DNA distribution in human cell cultures and that diploid cells such as WI‐38 may be more appropriate for this purpose.
Selection of optical filters for laser flow cytometry using fluorescent conjugates
Authors: Martin C. Leon , Rolland Jennifer M. , Nairn Richard C. , Muller H. Konrad
Published: June 2010

High performance optical filters improve sensitivity of detection and resolution of fluorescence in flow cytofluorimetry. Optimal optical barrier filters for laser‐illuminated flow cytofluorimeters differ from those for fluorescence microscopes with wide spectrum illumination. Filters for laser excitation must stop intense illumination at particular wavelengths while transmitting fluorescence emission maximally.

Using a Becton, Dickinson Fluorescence Activated Cell Sorter (FACS II) (Becton, Dickinson and Co., Sunnyvale, CA) a range of optical filters were compared for transmission characteristics and performance in fluorescence analysis of cells stained with fluorescein‐ or rhodamine‐labeled antibodies and of green and red fluorescent beads. Interference filters were generally superior to dyed glass filters: the Ortho 528 nm band pass (BP) filter (Ortho Instruments, Westwood, MA) (originally a Schott and Genossen product) was excellent for fluorescein analysis using the 488 nm laser line. In the absence of satisfactory interference filters, a Schott and Genossen 550 nm long pass (LP) filter (Schott America, NY) (dyed epoxy between two glass plates) was best for rhodamine analysis using the 514.5 nm line. Intrinsic fluorescence of some dyed glass filters rendered them unsuitable unless used in a prefiltered light path. Simultaneous analysis using two fluorochromes with overlapping excitation and emission spectra, e.g. fluorescein and rhodamine, will be suboptimal even with electronic dual fluorescence compensation; optimal analysis requires dual laser excitation with geometric separation of the light paths. For single laser systems, sensitivity and resolution are improved by using fluorochromes with fullyseparated emission spectra.
A microsample collection device for electrostatically sorted cells or particles and its preparative use for biochemical analysis
Authors: Hinson W. G. , Pipkin J. L. , Hudson J. L. , Anson J. F. , Tyrer H.
Published: June 2010

Biochemical analysis of proteins extracted from mammalian cell nuclei which were sorted according to DNA content was greatly simplified using a special collection device to recover sorted nuclei. The device was developed for use with a sorting flow cytometer to provide sufficient numbers of particles to achieve adequate sensitivity and resolution while minimizing sample degradation and loss. The design, evaluation and application of the collection device were described with emphasis on sample preparation.

Nuclear populations G0 + G1, S, and G2 + M, were sorted from regenerating rat liver tissue preparations and collected separately in the special chambers. Nuclear proteins were extracted and analyzed by polyacrylamide gel isoelectric focusing or capillary isotachophoresis.
Rapid isolation of cloned isotype switch variants using fluorescence activated cell sorting
Authors: Dangl Jeffery L. , Parks David R. , Oi Vernon T. , Herzenberg Leonard A.
Published: June 2010

We have used highly specific, directly fluorescein‐conjugated heterologous (conventional) and monoclonal antibodies directed against mouse immunoglobulin isotypes in conjunction with the fluorescence activated cell sorter (FACS) to enrich and clone hybridoma cells producing new immunoglobulin heavy chain constant regions. Each variant retains the parental heavy chain variable region and the parental immunoglobulin light chain; thereby each variant binds the same dansyl (DNS) hapten. These isotype switch variants occur at frequencies of approximately 10−5 to 10−6. We were able to isolate the variants by first sorting for an approximate 1000‐fold enrichment of the desired immunoglobulin‐producing cells, growing these cells for five to nine days, followed by a second 1000‐fold enrichment and direct cell cloning into 96 well culture trays. Clones were screened only 3–5 weeks after the original selection for secretion of dansyl‐binding immunoglobulin of the selected isotype. Judicious combination of existing methods permits improved analytical techniques using the cell sorter. These include: first, “red” fluorescence staining of dead cells with ethidium bromide or propidium iodide and using the red fluorescence measurement to exclude dead cells from the green fluorescence selection; and second, the use logarithmic amplification of fluorescence signals, allowing for more succinct selection of fluorescence parameters for sorting.
Cell cycle and 5′‐nucleotide phosphodiesterase activities in rat liver
Authors: Lo K. W. , Tsou K. C.
Published: June 2010

The postnatal developments of liver and serum 5′‐nucleotide phosphodiesterase (5′‐NPD) in Fischer 344 rats were studied. The liver enzyme activities were found to correlate well with the growth of the liver as measured by the wet weight and percentages of liver cells in the G2/M phases of the cell cycle as determined by flow cytofluorometry. Therefore, this enzyme may be intimately related to the growth of the liver.

Although the specific activities of the serum enzyme are three orders of magnitude less than that of the liver enzyme, very good correlation was obtained between the total serum and liver enzyme activities. This finding, therefore, suggests that the main origin of the serum enzyme is from liver. Multiple isozymes were detected in both liver and serum 5′‐nucleotide phosphodiesterase after polyacrylamide gel electrophoresis.
Cell cycle analysis in a human cell line (EUE cells)
Authors: Zippel R. , Martegani E. , Vanoni M. , Mazzini G. , Alberghina L.
Published: June 2010

A new procedure to determine the durations of cell cycle phases is based on the autoradiography of labeled nuclei and a standard chemical determination of DNA content per cell. It has been used to study the cell cycle timing of a human established cell line, EUE cells, so far poorly characterized in its kinetic aspects. The cycle phase durations obtained with this method are compared with the DNA frequency profiles determined by flow cytometry and found to agree closely. The relevance of the growth conditions of the exponentially growing cultures for a correct estimation of the cycle phase duration is indicated. Cell size distribution has been shown to vary according to growth condition.
Early stimulation, parental behavior, and the temperature of infant mice
Authors: Barnett S. A. , Walker K. Z.
Published: June 2010

The effects of early conditions of rearing on parental attention and on the heat exchanges of infant mice were examined: (1) Inbred migce 6 days old were exposed at 7.5 °C and returned to the nest. Mice which were rewarmed before return recieved more parental attention then cold mice or handled Controls. (2) Outbred mice 6 days old with injured ears were licked more by there mothers then uninjured litter mates. (3) Liking of genitalia alos occured but was unrelated to ear injury. (4) surface temperature of 6‐day old inbred mice declined at after artifical “licking” with a wet brush. (5) Surfarce temperature of inbred mice exposed at 22–28 °C for 2.5 min. declined at age 0–11 days and increased at age 15–20 days. Exposure at 15°C led to a declined in surface temperature at all ages. (6) Wild or laboratory mice 4 or 6 satge old were observe in their nests. Surface temperature decreased after parental licking or loss of contact with the litter and increased on contact with mother. Temperatures also increased rapidly if the mouse was held on the experimentor's hand.

in work on the effects of early conditions of rearing, attention should be paid to the minute details of parental attention and the heat exchanges of the toung animals.
Inorganic Analogues of Graphene
Authors: Rao C. N. R. , Nag Angshuman
Published: June 2010

The discovery of graphene has aroused great interest in the properties and phenomena exhibited by two‐dimensional inorganic materials, especially when they comprise only a single, two or a few layers. Graphene‐like MoS2 and WS2 have been prepared by chemical methods, and the materials have been characterized by electron microscopy, atomic force microscopy (AFM) and other methods. Boron nitride analogues of graphene have been obtained by a simple chemical procedure starting with boric acid and urea and have been characterized by various techniques that include surface area measurements. A new layered material with the composition BCN possessing a few layers and a large surface area discovered recently exhibits a large uptake of CO2.
Platinum‐Catalyzed Regioselective Formation of β‐Alkoxy Ketones from Internal Alkynes
Authors: Yang Daxin , Huang Jianfeng , Liu Bo
Published: June 2010

A catalytic amount of Zeise's dimer and 15‐crown‐5 were combined to effectively promote the regioselective formation of β‐alkoxy ketones from 2‐(homopropargyloxy)ethanols in dimethoxyethane. The desired products were obtained in 59–98 % yields with up to 17:1 exo/endo regioselectivity. The methodology allows access to β‐hydroxy ketones as an aldol reaction alternative.
Analyses of sulfonamide antibiotics by a successive anion‐ and cation‐selective injection coupled to microemulsion electrokinetic chromatography
Authors: Lin Yun‐Ta , Liu Yu‐Wei , Cheng Yi‐Jie , Huang Hsi‐Ya
Published: June 2010

In this study, an MEEKC was used to detect and analyze nine sulfonamide antibiotics. Owing to an insufficient sensitivity of on‐column UV detection, a field‐amplified sample injection, successive anion‐ and cation‐selective injection, was used for the on‐line concentration of the nine antibiotics. In the successive anion‐ and cation‐selective injection mode, a leading water plug was introduced prior to anion injection, and then an acidic plug followed by a terminal water plug had to be used before subsequent cation injection. The results indicated some sulfonamides (sulfamonomethoxine, sulfamethazine, sulfamerazine and sulfadiazine) were determined as split signals in pairs, and this was likely due to the use of a longer acid plug (360 s) which caused the sulfonamide anions and cations to be stacked in two distinct zones of the leading water and acid plugs. Meanwhile, all the sulfonamides that were introduced either by anion or cation injection were stacked within the leading water plug when a shorter acid plug (210 s) was used. As a result, the nine sulfonamides were determined as single and symmetrical peaks with low LODs (0.9–4.2 μg/L). Furthermore, the MEEKC method was successfully applied for the detection of trace sulfonamide residues in several food and water samples.
DNA cross‐link induced by trans‐4‐hydroxynonenal
Authors: Huang Hai , Kozekov Ivan D. , Kozekova Albena , Wang Hao , Lloyd R. Stephen , Rizzo Carmelo J. , Stone Michael P.
Published: June 2010

Trans−4‐Hydroxynonenal (HNE) is a peroxidation product of ω−6 polyunsaturated fatty acids. Michael addition of HNE to deoxyguanosine yields four diastereomeric 1,N2‐dG adducts. The adduct of (6S,8R,11S) stereochemistry forms interstrand N2‐dG:N2‐dG cross‐links in the 5′‐CpG‐3′ sequence. It has been compared with the (6R,8S,11R) adduct, incorporated into 5′‐d(GCTAGCXAGTCC)‐3' · 5'‐d(GGACTCGCTAGC)‐3′, containing the 5′‐CpG‐3′ sequence (X = HNE‐dG). Both adducts rearrange in DNA to N2‐dG aldehydes. These aldehydes exist in equilibrium with diastereomeric cyclic hemiacetals, in which the latter predominate at equilibrium. These cyclic hemiacetals mask the aldehydes, explaining why DNA cross‐linking is slow compared to related 1,N2‐dG adducts formed by acrolein and crotonaldehyde. Both the (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals are located within the minor groove. However, the (6S,8R,11S) cyclic hemiacetal orients in the 5′‐direction, while the (6R,8S,11R) cyclic hemiacetal orients in the 3′‐direction. The conformations of the diastereomeric N2‐dG aldehydes, which are the reactive species involved in DNA cross‐link formation, have been calculated using molecular mechanics methods. The (6S,8R,11S) aldehyde orients in the 5′‐direction, while the (6R,8S,11R) aldehyde orients in the 3′‐direction. This suggests a kinetic basis to explain, in part, why the (6S,8R,11S) HNE adduct forms interchain cross‐links in the 5′‐CpG‐3′ sequence, whereas (6R,8S,11R) HNE adduct does not. The presence of these cross‐links in vivo is anticipated to interfere with DNA replication and transcription, thereby contributing to the etiology of human disease. Environ. Mol. Mutagen., 2010. © 2010 Wiley‐Liss, Inc.
Patterns of grain‐size dependent sediment transport in low‐ordered, ephemeral channels
Authors: Yuill Brendan T. , Nichols Mary H.
Published: June 2010

Sediment data were analyzed to determine grain‐size dependant factors affecting sediment transport in a low‐ordered, ephemeral watershed. Sediment and flow samples were collected during 22 flow events at the outlet of a 4·53 ha sub‐watershed within the Walnut Gulch Experimental Watershed in south‐eastern Arizona. Measured concentrations ranged from 4191 to 115 045 mg l−1 and included grain sizes up to 8·0 mm in diameter. Two grain‐size dependent transport patterns were observed, that of the finer grain‐size fraction (approximately < 0·25 mm) and that of a coarser grain‐size fraction (approximately ≥ 0·25 mm). The concentration of the fine fraction decreased with flow duration, peaking near the beginning of a flow event and declining thereafter. The concentration of the fine fraction showed slight trends with season and recovery period. The concentration of the coarse fraction displayed a slight negative trend with instantaneous discharge and was not correlated with event duration. These patterns typically produced a condition where the majority of the fine fraction of the sediment yield was evacuated out of the watershed before the hydrograph peak while the majority of the coarser sediment was evacuated during the falling limb of the hydrograph. Each grain‐size dependent transport pattern was likely influenced by the source of the associated sediment. At the flow event time scale, the fines were primarily wash load, supplied from the hillslopes and the coarser grains were entrained from the channel bed. Because transport patterns differ based on grain size, attempts to define the total sediment concentration and sediment yield by the behavior of a single grain‐size fraction may lead to erroneous results, especially when a large range of sediment grain sizes are present. Copyright © 2010 John Wiley & Sons, Ltd.
Axonal integrity in the absence of functional peroxisomes from projection neurons and astrocytes
Authors: Bottelbergs Astrid , Verheijden Simon , Hulshagen Leen , Gutmann David H. , Goebbels Sandra , Nave Klaus‐Armin , Kassmann Celia , Baes Myriam
Published: June 2010

Ablation of functional peroxisomes from all neural cells in Nestin‐Pex5 knockout mice caused remarkable neurological abnormalities including motoric and cognitive malfunctioning accompanied by demyelination, axonal degeneration, and gliosis. An oligodendrocyte selective Cnp‐Pex5 knockout mouse model shows a similar pathology, but with later onset and slower progression. Until now, the link between these neurological anomalies and the known metabolic alterations, namely the accumulation of very long‐chain fatty acids (VLCFA) and reduction of plasmalogens, has not been established. We now focused on the role of peroxisomes in neurons and astrocytes. A neuron‐specific peroxisome knockout model, NEX‐Pex5, showed neither microscopic nor metabolic abnormalities indicating that the lack of functional peroxisomes within neurons does not cause axonal damage. Axonal integrity and normal behavior was also preserved when peroxisomes were deleted from astrocytes in GFAP‐Pex5−/− mice. Nevertheless, peroxisomal metabolites were dysregulated in brain including a marked accumulation of VLCFA and a slight reduction in plasmalogens. Interestingly, despite minor targeting of oligodendrocytes in GFAP‐Pex5−/− mice, these metabolic perturbations were also present in isolated myelin indicating that peroxisomal metabolites are shuttled between different brain cell types. We conclude that absence of peroxisomal metabolism in neurons and astrocytes does not provoke the neurodegenerative phenotype observed after deleting peroxisomes from oligodendrocytes. Lack of peroxisomal metabolism in astrocytes causes increased VLCFA levels in myelin, but this has no major impact on neurological functioning. © 2010 Wiley‐Liss, Inc.
Organization of glial cells in the adult sea cucumber central nervous system
Authors: Mashanov Vladimir S. , Zueva Olga R. , Garcia‐Arraras Jose E.
Published: June 2010

The nervous system of echinoderms has long been considered too unique to be directly comparable to the nervous system of other Deuterostomia. Using two novel monoclonal antibodies in combination with epifluorescence, confocal, and electron microscopy, we demonstrate here that the central nervous system of the sea cucumber Holothuria glaberrima possesses a major non‐neuronal cell type, which shares striking similarities with the radial glia of chordates. The basic features in common include (a) an elongated shape, (b) long radial processes, (c) short lateral protrusions branching off the main processes and penetrating into the surrounding neuropile, (d) prominent orderly oriented bundles of intermediate filaments, and (e) ability to produce Reissner's substance. Radial glia account for the majority of glia cells in echinoderms and constitutes more than half of the total cell population in the radial nerve cord and about 45% in the circumoral nerve ring. The difference in glia cell number between those regions is significant, suggesting structural specialization within the seemingly simple echinoderm nervous system. Both cell death and proliferation are seen under normal physiological conditions. Although both glia and neurons undergo apoptosis, most of the mitotic cells are identified as radial glia, indicating a key role of this cell type in cell turnover in the nervous system. A hypothesis is proposed that the radial glia could be an ancestral feature of the deuterostome nervous system, and the origin of this cell type might have predated the diversification of the Chordata and Ambulacraria lineages. © 2010 Wiley‐Liss, Inc.
Dynamic expression of Cx47 in mouse brain development and in the cuprizone model of myelin plasticity
Authors: Parenti Rosalba , Cicirata Federico , Zappalà Agata , Catania Angela , La Delia Francesco , Cicirata Valentina , Tress Oliver , Willecke Klaus
Published: June 2010

The study shows the dynamic expression of connexin47 (Cx47) in oligodendrocytes and myelin of mice, either in myelinogenesis occurring in early development or in an experimental model of new‐myelinogenesis of adult mice. Cx47 first appeared in the embryonic mouse brain at E10.5 successively the expression increased, principally in regions populated by developing oligodendrocytes. The expression declined postnatally toward adulthood and immunoreactivity was restricted to a few specific areas, such as the corpus callosum, the striatum, the cerebellum, and the spinal cord. Since the expression of Cx47 in developing oligodendrocytes preceded those of Cx32 and Cx29, a role of Cx47 in myelinogenesis was postulated. This hypothesis was tested in a model of re‐myelination, which principally involved the corpus callosum, occurring in adult mice by treatment with cuprizone. Cx47 was upregulated during demyelination and recovered during the remyelination phase. During demyelination, Cx47 was first over‐expressed in the corpus callosum and later, when the myelin virtually disappeared in the injured areas, Cx47 was expressed in astrocytes located inside and closely around the demyelinated areas. The remyelination of injured areas occurred after stopping the administration of cuprizone and continued to complete recovery. In this period the expression of Cx47 shifted from astrocytes to newly‐formed myelin. Thus, Cx47 exhibits in this model a transient and de novo expression in astrocytes with a topographic segregation in the injured areas, only when oligodendrocytes and the myelin were most severely affected. Taken as a whole the evidence suggests that Cx47 play a key role in myelination. © 2010 Wiley‐Liss, Inc.
Toll‐like receptor expression in the peripheral nerve
Authors: Goethals Sofie , Ydens Elke , Timmerman Vincent , Janssens Sophie
Published: June 2010

Toll‐like receptors comprise a family of evolutionary conserved pattern recognition receptors that act as a first defense line in the innate immune system. Upon stimulation with microbial ligands, they orchestrate the induction of a host defense response by activating different signaling cascades. Interestingly, they appear to detect the presence of endogenous signals of danger as well and as such, neurodegeneration is thought to trigger an immune response through ligation of TLRs. Though recent data report the expression of various TLRs in the central nervous system, TLR expression patterns in the peripheral nervous system have not been determined yet. We observed that Schwann cells express relatively high levels of TLRs, with especially TLR3 and TLR4 being prominent. Sensory and motor neurons hardly express TLRs at all. Through the use of NF‐κB signaling as read‐out, we could show that all TLRs are functional in Schwann cells and that bacterial lipoprotein, a ligand for TLR1/TLR2 receptors yields the strongest response. In sciatic nerve, basal levels of TLRs closely reflect the expression patterns as determined in Schwann cells. TLR3, TLR4, and TLR7 are majorly expressed, pointing to their possible role in immune surveillance. Upon axotomy, TLR1 becomes strongly induced, while most other TLR expression levels remain unaffected. Altogether, our data suggest that similar to microglia in the brain, Schwann cells might act as sentinel cells in the PNS. Furthermore, acute neurodegeneration induces a shift in TLR expression pattern, most likely illustrating specialized functions of TLRs in basal versus activated conditions of the peripheral nerve. © 2010 Wiley‐Liss, Inc.
Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma: A Weighty Connection
Authors: Starley Brad Q. , Calcagno Christopher J. , Harrison Stephen A.
Published: June 2010

Hepatocellular carcinoma (HCC) is a common and deadly malignancy that is increasing in incidence in developed countries. The emergence of hepatitis C virus (HCV) accounts for about half of this increase in HCC, although the etiology of HCC in 15%-50% of new HCC cases remains unclear. The most common form of chronic liver disease in developed countries is nonalcoholic fatty liver disease (NAFLD), which encompasses a broad spectrum of histopathology. The prevalence of NAFLD, including the more aggressive nonalcoholic steatohepatitis (NASH), is increasing with the growing epidemics of diabetes and obesity. NASH can progress to cirrhosis and its related complications. Growing evidence suggests that NASH accounts for a large proportion of idiopathic or cryptogenic cirrhosis, which is associated with the typical risk factors for NASH. HCC is a rare, although important complication of NAFLD. Diabetes and obesity have been established as independent risk factors for the development of HCC. New evidence also suggests that hepatic iron deposition increases the risk of HCC in NASH-derived cirrhosis. Multiple case reports and case reviews of HCC in the setting of NASH support the associations of diabetes and obesity with the risk of HCC, as well as suggest age and advanced fibrosis as significant risks. Insulin resistance and its subsequent inflammatory cascade that is associated with the development of NASH appear to play a significant role in the carcinogenesis of HCC. The complications of NASH, including cirrhosis and HCC, are expected to increase with the growing epidemic of diabetes and obesity. (Hepatology 2010;51:1820–1832)
Blood Lactate As A Prognostic Marker in Acetaminophen–Induced Acute Liver Failure
Authors: Schmidt Lars E. , Larsen Fin Stolze
Published: June 2010

Abstract

Background:

Although the King's College Hospital (KCH) selection criteria for emergency liver transplantation in paracetamol–induced acute liver failure are widely used, strategies to improve sensitivity and facilitate earlier transplantation are required. We investigated the use of arterial blood lactate measurement for the identification of transplantation candidates.

Methods:

In a single–centre study, we measured arterial blood lactate early (median 4 h) and after fluid resuscitation (median 12 h) in patients admitted to a tertiary–referral intensive–care unit. Threshold values that best identified individuals likely to die without liver transplantation were derived in a retrospective initial sample of 103 patients with paracetamol–induced acute liver failure and applied to a prospective validation sample of 107 patients. Predictive value and speed of identification were compared to those of the KCH criteria.

Findings:

In the initial sample, median lactate was significantly higher in non–surviving patients than in survivors both in the early samples (8.5 [range 1.7–21.0] vs. 1.4 [0.53–7.9] mmol/L, p<0.0001) and after fluid resuscitation (5.5 [1.3–18.6] vs. 1.3 [0.26–3.2], p<0.0001). Applied to the validation sample, a threshold value of 3.5 mmol/L early after admission had sensitivity 67%, specificity 95%, positive likelihood ratio 13, and negative likelihood ratio 0.35; the corresponding values for a threshold of 3.0 mmol/L after fluid resuscitation were 76%, 97%, 30, and 0.24. Combined early and postresuscitation lactate concentrations had similar predictive ability to KCH criteria but identified non–surviving patients earlier (4 [3–13] vs 10 [3.5–19.5] h, p=0.01). Addition of postresuscitation lactate concentration to KCH criteria increased sensitivity from 76% to 91% and lowered negative likelihood ratio from 0.25 to 0.10.

Interpretation:

Arterial blood lactate measurement rapidly and accurately identifies patients who will die from paracetamol–induced acute liver failure. Its use could improve the speed and accuracy of selection of appropriate candidates for transplantation.
New Ways of Interfering With Hcv Replication
Authors: Hasselblatt Peter , Blum Hubert E. , Offensperger Wolf-Bernhard
Published: June 2010

RNA interference is a cellular process of gene silencing in which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases. The introduction of approximately 22–nt small interfering RNAs (siRNAs) into mammalian cells can specifically silence cellular mRNAs without induction of the nonspecific IFN responses that are activated by longer RNA duplexes. We investigate in this article whether siRNAs can also silence the expression of the cytoplasmically replicating hepatitis C virus (HCV) RNAs by using a replicon system that supports robust HCV replication, but not the production of infectious virions. We report the efficient silencing of both cellular lamin AC and HCV RNAs in Huh–7 hepatoma cell lines supporting HCV replication. Silencing of HCV RNAs was dose dependent and specific, inasmuch as two HCV variants that differ by 3 nt within the target sequence were only silenced by the exact homologous sequence for each. siRNAs designed to target HCV RNA triggered an exponential decrease in HCV RNA, resulting in an 80–fold decrease in HCV RNA after 4 days. The introduction of siRNAs into cells with established HCV replication cured >98% of these cells of detectable HCV antigen and replication–competent HCV RNAs. These data support the principle of siRNA–based HCV antiviral therapy.
Car Unlikely to Significantly Modulate Acetaminophen Hepatotoxicity in Most Humans
Authors: Nelson Sidney D. , Slattery John T. , Thummel Kenneth E. , Watkins Paul B.
Published: June 2010

We have identified the xenobiotic receptor CAR (constitutive androstane receptor) as a key regulator of acetaminophen metabolism and hepatotoxicity. Known CAR activators as well as high doses of acetaminophen induced expression of three acetaminophen–metabolizing enzymes in wild–type but not in CAR null mice, and the CAR null mice were resistant to acetaminophen toxicity. Inhibition of CAR activity by administration of the inverse agonist ligand androstanol 1 hour after acetaminophen treatment blocked hepatotoxicity in wild–type but not CAR null mice. These results suggest an innovative therapeutic approach for treating the adverse effects of acetaminophen and potentially other hepatotoxic agents.
A Model for the Study of Hepatitis C Virus Entry
Authors: Castet Valérie , Moradpour Darius
Published: June 2010

The study of hepatitis C virus (HCV), a major cause of chronic liver disease, has been hampered by the lack of a cell culture system supporting its replication. Here, we have successfully generated infectious pseudo–particles that were assembled by displaying unmodified and functional HCV glycoproteins onto retroviral and lentiviral core particles. The presence of a green fluorescent protein marker gene packaged within these HCV pseudo–particles allowed reliable and fast determination of infectivity mediated by the HCV glycoproteins. Primary hepatocytes as well as hepato–carcinoma cells were found to be the major targets of infection in vitro. High infectivity of the pseudo–particles required both E1 and E2 HCV glycoproteins, and was neutralized by sera from HCV–infected patients and by some anti–E2 monoclonal antibodies. In addition, these pseudo–particles allowed investigation of the role of putative HCV receptors. Although our results tend to confirm their involvement, they provide evidence that neither LDLr nor CD81 is sufficient to mediate HCV cell entry. Altogether, these studies indicate that these pseudo–particles may mimic the early infection steps of parental HCV and will be suitable for the development of much needed new antiviral therapies.
GSK3β overexpression induces neuronal death and a depletion of the neurogenic niches in the dentate gyrus
Authors: Sirerol‐Piquer MaSalomé , Gomez‐Ramos Pilar , Hernández Félix , Perez Mar , Morán María A. , Fuster‐Matanzo Almudena , Lucas José J. , Avila Jesús , García‐Verdugo Jose Manuel
Published: June 2010

Overexpression of GSK3β in transgenic mice induces learning deficits and some features associated with Alzheimer's disease (AD), including dentate gyrus (DG) atrophy. Here, we assessed whether these mice also recapitulate DG atrophy as well as impaired neurogenesis reported in AD. Ultrastructural analysis revealed that there were fewer and more disorganized neurogenic niches in these animals, coupled with an increase in the proportion of immature neurons. Indeed, the maturation of granule cells is delayed as witnessed by the alterations to the length and patterning of their dendritic trees and to the mossy fiber terminals. Together with an increase in neuronal death, these phenomena lead to a marked decrease in the number and disorganization of granule cells of the DG. Our results suggest that GSK3β overexpression perturbs proliferation and maturation, resulting in the loss of immature neurons. In turn, the activation of microglia is stimulated in conjunction with a decrease in the birth of new functional neurons, leading to the deterioration of this structure. These data support the idea that by inducing degeneration of the DG, GSK3β could be involved in the pathogenesis of AD. © 2010 Wiley‐Liss, Inc.
Projections to the anterodorsal thalamus and lateral mammillary nuclei arise from different cell populations within the postsubiculum: Implications for the control of head direction cells
Authors: Yoder Ryan M. , Taube Jeffrey S.
Published: June 2010

The neural representation of directional heading is encoded by a population of cells located in a circuit that includes the postsubiculum (PoS), anterodorsal thalamus (ADN), and lateral mammillary nuclei (LMN). Throughout this circuit, many cells rely on both movement‐ and landmark‐related information to discharge as a function of the animal's directional heading. The PoS projects to both the ADN and LMN, and these connections may convey critical spatial information about landmarks, because lesions of the PoS disrupt landmark control in head direction (HD) cells and hippocampal place cells [Goodridge and Taube (1997) J Neurosci 17:9315–9330; Calton et al. (2003) J Neurosci 23:9719–9731]. The PoS → ADN projection originates in the deep layers of PoS, but no studies have determined whether the PoS → LMN projection originates from the same cells that project to ADN. To address this issue, two distinct cholera toxin‐subunit B (CTB) fluorophore conjugates (Alexa Fluor 488 and Alexa Fluor 594) were injected into the LMN and ADN of the same rats, and PoS sections were examined for cell bodies containing either or both CTB conjugates. Results indicated that the PoS → LMN projection originates exclusively from a thin layer of cells located superficial to the layer(s) of PoS → ADN projection cells, with no overlap. To verify the laminar distribution and morphological characteristics of PoS → LMN and PoS → ADN cells, biotinylated dextran amine was injected into LMN or ADN of different rats, and tissue sections were counterstained with thionin. Results indicated that the PoS → LMN projection arises from large pyramidal cells in layer IV, whereas the PoS → ADN projection arises from a heterogeneous cell population in layers V/VI. This study provides the first evidence that the PoS → ADN and PoS → LMN projections arise from distinct, nonoverlapping cell layers in PoS. Functionally, the PoS may provide landmark information to HD cells in LMN. © 2010 Wiley‐Liss, Inc.
Behavior abnormality following intravenous immunoglobulin treatment in patients with primary antibody deficiencies
Authors: Saroukhani Sepide , Aghamohammadi Asghar , Mahmoudi‐Gharaei Javad , Abolhassani Hassan , Cheraghi Taher , Imanzadeh Amir , Moazzami Kasra , Parvaneh Nima , Rezaei Nima
Published: June 2010

Objective

Treatment with intravenous immunoglobulin (IVIG) is considered a safe therapy for patients with primary antibody deficiencies (PADs), whilst adverse effects have been frequently reported. Meantime behavioral disorders reactions have not been reported yet. In this study, we describe for the first time a group of patients with PADs, who were under IVIG therapy and experienced some behavioral disorders.

Methods

Five patients, including two hyper IgM syndromes, one X‐linked agammaglobulinemia, one common variable immunodeficiency, and one hypo IgM disease, were surveyed. Analysis of Conner's Parents Rating Scales‐Revised Short (CPRS‐R:S) and child behavior checklist (CBCL) was performed for the patients, suspected to hyperactivity.

Results

Analysis of CPRS‐R:S showed an evidence of mild hyperactivity before IVIG administration in four patients, whereas another patient had evidence of severe hyperactivity. After IVIG administration, hyperactivity scores of three patients were changed from mild hyperactive behavior to markedly hyperactive behavior or attention deficit hyperactivity disorder range of hyperactivity. In the CBCL scores, there were abnormal externalization scores for three patients; while two remaining patients had abnormal internalization scores.

Conclusions

Although predisposition to behavioral disorders can be due to a genetic background, further investigations are necessary to test the hypotheses about responsibility of either IVIG or underling disease in progression of behavioral abnormalities. Copyright © 2010 John Wiley & Sons, Ltd.
Suicide inactivation of the diphenolase and monophenolase activities of tyrosinase
Authors: Muñoz‐Muñoz Jose Luis , Garcia‐Molina Francisco , Varon Ramon , Garcia‐Ruíz Pedro A. , Tudela Jose , Garcia‐Cánovas Francisco , Rodríguez‐López Jose Neptuno
Published: June 2010

The suicide inactivation mechanism of tyrosinase acting on its phenolic substrates has been studied. Kinetic analysis of the proposed mechanism during the transition phase provides explicit analytical expressions for the concentrations of o‐quinone versus time. The electronic, steric, and hydrophobic effects of the phenolic substrates influence the enzymatic reaction, increasing the catalytic speed by three orders of magnitude and the inactivation by one order of magnitude. To explain this suicide inactivation, we propose a mechanism in which the enzymatic form oxy‐tyrosinase is responsible for the inactivation. In this mechanism, the rate constant of the reaction would be directly related with the strength of the nucleophilic attack of the C‐1 hydroxyl group, which depends on the chemical shift of the carbon C‐1 (δ1) obtained by 13C‐NMR. The suicide inactivation would occur if the C‐2 hydroxyl group transferred the proton to the protonated peroxide, which would again act as a general base. In this case, the coplanarity between the copper atom, the oxygen of the C‐1 and the ring would only permit the oxidation/reduction of one copper atom, giving rise to copper (0), hydrogen peroxide, and an o‐quinone, which would be released, thus inactivating the enzyme. One possible application of this property could be the use of these suicide substrates as skin depigmenting agents. © 2010 IUBMB IUBMB Life 62(7): 539–547, 2010
The mitochondrial bioenergetic capacity of carcinomas
Authors: Formentini Laura , Martínez‐Reyes Inmaculada , Cuezva José M.
Published: June 2010

Metabolic reprogramming of cancer cells is a phenotypic trait necessary to promote proliferation and survival. Despite past controversies, recent transcriptomic, proteomic, functional and structural studies of mitochondria of the cancer cell indicate that an impaired biogenesis and activity of the organelle is required for the development of some tumors. Cancer aggressiveness can be estimated by its bioenergetic signature, a protein ratio that correlates the expression of β‐F1‐ATPase of oxidative phosphorylation relative to the glycolytic GAPDH. The bioenergetic signature also provides a gauge that informs of the metabolic activity of tumors and cancer cells as well as of the response to chemotherapy. The convergence of different epithelial tumors on the same bioenergetic signature supports that it provides an important tool and common target for cancer therapy. We stress that targeting the energetic metabolism of tumors affords a valuable strategy to combat the disease. © 2010 IUBMB IUBMB Life 62(7): 554–560, 2010
The polarity of the amino acid residue 118 of calcineurin B is closely linked to calcineurin enzyme activity
Authors: Chen Qing , Wu Wu , Li Jing , Wei Qun
Published: June 2010

Calcineurin(Cn), a multifunctional regulator expressed in several tissues and organs, consists of CnA (catalytic subunit) and CnB (regulatory subunit). The crystal structure shows that the hydrophobic groove formed by 118–123 residues of CnB is necessary for its interactions with two different immunosuppressant‐immunophilin complexes and with CnA. In this report, we focus on Met118 of CnB to study the association between conformational states of CnB and the phosphatase activity of Cn. We found that hydrophobicity in the region around site118 of CnB is essential for the Cn activity. Polar mutants significantly weakened the enzymatic activity compared with the nonpolar ones. The data showed that some modest alterations in the vicinity of site118 impaired the integrality and compactness of hydrophobic microenvironment, and this might explain why CnB mutants defective in hydrophobicity failed in activating Cn. This requirement of hydrophobic microenvironment around site118 in CnB suggests that, besides the mutations in the catalytic subunit CnA, which impairs Cn phosphatase activity, and had been identified to be associated with diseases such as Alzheimer's disease (AD), the mutations in CnB might also affect Cn enzymatic activityin vivo, and this might be helpful for our further research on mechanisms of diseases associated with Cn. © 2010 IUBMB IUBMB Life 62(7): 561–567, 2010
FT‐IR spectroscopy in diagnosis of diabetes in rat animal model
Authors: Severcan Feride , Bozkurt Ozlem , Gurbanov Rafig , Gorgulu Guvenc
Published: June 2010

In recent years, Fourier Transform Infrared (FT‐IR) spectroscopy has had an increasingly important role in the field of pathology and diagnosis of disease states. In the current study, FT‐IR spectroscopy together with cluster analysis were used as a diagnostic tool in the discrimination of diabetic samples from control ones in rat kidney plasma membrane apical sides (brush‐border membranes), liver microsomal membranes and Extensor digitorum longus (EDL) and Soleus (SOL) skeletal muscle tissues.

A variety of alterations in the spectral parameters, such as frequency and signal intensity/area was observed in diabetic tissues and membranes compared to the control samples. Based on these spectral variations, using cluster analysis successful differentiation between diabetic and control groups was obtained in different spectral regions. The results of this current study further revealed the power and sensitivity of FT‐IR spectroscopy in precise and automated diagnosis of diabetes. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Image enhancement in ultramicroscopy by improved laser light sheets
Authors: Saghafi Saiedeh , Becker Klaus , Jährling Nina , Richter Melanie , Kramer Edgar R. , Dodt Hans‐Ulrich
Published: June 2010

In the majority of implementations of light sheet microscopy, such as ultramicroscopy, the laser beam illuminating the specimen is truncated by a slit aperture before it is focused to a light sheet by a single cylindrical lens. A light sheet generated in this way can be made very thin near to the focal point, but unfortunately its Rayleigh range is severely limited. This problem can be partially solved by using a smaller slit aperture. However, this also causes a major loss in power, a severe broadening of the beam waist, and thus a significant loss of resolution along the detection axis. We developed improved light‐sheet‐generation optics, which provide longer Raleigh ranges, whilst retaining beam waists comparable to our standard system with one cylindrical lens. Using the modified system we achieved a marked improvement in the resolution of ultramicroscopy reconstructions of representative biological specimens. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Fish consumption, bone mineral density, and risk of hip fracture among older adults: The cardiovascular health study
Authors: Virtanen Jyrki K , Mozaffarian Dariush , Cauley Jane A , Mukamal Kenneth J , Robbins John , Siscovick David S
Published: June 2010

Marine n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may be beneficial for bone health, but few studies have investigated the association with fish consumption. Our aim was to study associations of fish and EPA + DHA consumption with bone mineral density (BMD) and hip fracture risk and determine whether high linoleic acid (LA) intake, the major dietary n-6 PUFA, modifies the associations. The study population consisted of 5045 participants aged 65 years and older from the Cardiovascular Health Study. Data on BMD were available for 1305 participants. Food-frequency questionnaire was used to assess dietary intake, and hip fracture incidence was assessed prospectively by review of hospitalization records. After multivariable adjustment, femoral neck BMD was 0.01 g/cm2 lower in the highest versus lowest tuna/other-fish intake category (p = .05 for trend). EPA + DHA intake (higher versus lower median of 0.32 g/day) was associated with lower femoral neck BMD (0.66 versus 0.71 g/cm2, p < .001) among those with LA intake greater than the median 12.1 g/day (p = .03 for interaction). No significant associations were found with total-hip BMD. During mean follow-up of 11.1 years, 505 hip fractures occurred. Fish or EPA + DHA consumption was not significantly associated with fracture incidence [hazard ratio (HR) for extreme categories: HR = 1.23, 95% confidence interval (CI) 0.83–1.84 for tuna/other fish; HR = 1.16, 95% CI 0.91–1.49 for fried fish; and HR = 0.98, 95% CI 0.71–1.36 for EPA + DHA]. High LA intake did not modify these associations. In this large prospective cohort of older adults, fish consumption was associated with very small differences in BMD and had no association with hip fracture risk. © 2010 American Society for Bone and Mineral Research.
Problems after discharge and understanding of communication with their primary care physicians among hospitalized seniors: A mixed methods study
Authors: Arora Vineet M. , Prochaska Megan L. , Farnan Jeanne M. , D'Arcy V Michael J. , Schwanz Korry J. , Vinci Lisa M. , Davis Andrew M. , Meltzer David O. , Johnson Julie K.
Published: June 2010

BACKGROUND:

Communication and coordination with primary care physicians (PCPs) is recommended to ensure safe care transitions for hospitalized older patients. Understanding patient experiences of problems after discharge can help clinical teams design more patient‐centered care transitions.

OBJECTIVE:

To report older patients' experiences with problems after hospital discharge and investigate whether PCPs were aware of their hospitalization.

DESIGN:

Prospective mixed methods study.

SETTING:

Single academic medical center.

PATIENTS:

Hospitalized patients and PCPs.

MEASUREMENTS:

Telephone interviews of frail, older general medical patients conducted 2 weeks after discharge to elicit patient problems after discharge, such as: (1) obtaining medications, or follow‐up appointments; and (2) perceptions of hospital physician communication with their PCP. For each patient interviewed, their PCP was faxed a survey 2 weeks after discharge to assess awareness of hospitalization.

RESULTS:

Forty‐two percent (27) of patients reported 42 different post‐discharge problems. The most frequently reported problems were difficulty with follow‐up appointments or tests (12). Other reported problems included readmission and return to the Emergency Department (10), problems with medications (8), not‐prepared for discharge (8), and hospital complications or questions (4). Thirty percent of PCPs were unaware of patient hospitalization. Patients were twice as likely to report a problem if their PCP was unaware of the hospitalization (31% PCP aware, vs. 67% PCP not aware; P < 0.05).

CONCLUSION:

This study suggests that many frail, older patients reported problems after discharge and were twice as likely to do so when the patient's PCP was not aware of the hospitalization. Systematic interventions to improve communication with PCPs during patient hospitalization are needed. Journal of Hospital Medicine 2010. © 2010 Society of Hospital Medicine.