bluuebunny/crossref_metadata_2025 huggingface dataset:
Hornet Flight Activity and its Correlation with UVB Radiation, Temperature and Relative Humidity
Authors: Volynchik Stanislav , Plotkin Marian , Bergman David J. , Ishay Jacob S.
Published: September 2007

During the active season, extending from June to October, hornets emerge from their nest in the field in all the daytime hours. In the beginning of the season, when the number of workers is relatively small, the number of exits from the nest is fairly uniform numerically throughout the day. However, with the increase in hornet population from July onwards, the number of workers emerging from the nest entrance around noon (1100–1300 h) is by 1–2 orders of magnitude greater than the number of those emerging in the morning or evening hours. This disparity persists till September or October, at which time the workers revert to behave as in the beginning of the season. It appears, therefore, that in this period hornet activities outside the nest are coordinated with the meteorological conditions, and in this regard, the highest correlation is with the ultra violet B (UVB) radiation level and to a lesser extent with the temperature. Presumably, also, the greater noon‐hour activity in the nests of hornets in the field stems from the digging hornets benefiting from the greater availability of solar energy at noon, mainly that of UVB radiation. We assume that the hornets are able to utilize the UVB radiation, but what part of their body is “absorbing” the UVB energy is still a matter of further investigation.
Spatial Distribution of Protein Damage by Singlet Oxygen in Keratinocytes
Authors: He Yu‐Ying , Council Sarah E. , Feng Li , Bonini Marcelo G. , Chignell Colin F.
Published: September 2007

Singlet oxygen may be generated in cells by either endogenous or exogenous photosensitizers as a result of exposure to UV or visible irradiation. We have used immuno‐spin trapping (Free Radic. Biol. Med. 36: 1214, 2004) to identify the subcellular targets of singlet oxygen generated by rose bengal (RB). Confocal fluorescence microscopy of HaCaT keratinocytes incubated with RB clearly showed that the dye entered the cells and was located mainly in the perinuclear region, probably associated with the Golgi apparatus and endoplasmic reticulum. Previous studies by Wright et al. (Free Radic. Biol. Med.34: 637, 2003) have shown that long‐lived protein hydroperoxides (POOH) are present in cells exposed to singlet oxygen‐generating dyes. The addition of reducing metal ions such as Cu+ to POOH results in the generation of protein‐derived radicals, POO• and PO•, which react with the spin trap 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO) to give relatively stable spin adducts. In order to determine the subcellular localization of the protein‐DMPO adducts, we exposed keratinocytes to RB/light exposure and then incubated the cells with Cu+ and DMPO. After staining with antibody against DMPO followed by a secondary Alexa Fluor 488 goat anti‐rabbit IgG, the intracellular distribution of protein‐DMPO adducts was determined by confocal microscopy. The subcellular localization of the protein DMPO adducts was coincident with that of RB. This approach may provide information on the spatial distribution of singlet oxygen generated in cells.
The Solvent‐Initiated Photochemistry of Tetrachloropalladate(II) in Chloroform
Authors: Le Anh L. , Hoggard Patrick E.
Published: September 2007

Under 254 nm irradiation in chloroform, [PdCl4]2− is protonated to H2PdCl4 by HCl, generated through absorption of light by CHCl3. This stands in contrast to the behavior of [PtCl4]2− and [Pt(bpy)Cl2], which undergo photooxidation under the same conditions.
Sensitive Detection of PrPSc by Western Blot Assay Based on Streptomycin Sulphate Precipitation
Authors: Dong C.‐F. , Huang Y.‐X. , An R. , Chen J.‐M. , Wang X.‐F. , Shan B. , Lei Y.‐J. , Han L. , Zhang B.‐Y. , Han J. , Dong X.‐P.
Published: September 2007

Transmissible spongiform encephalopathies, also termed prion diseases, are fatal neurodegenerative disorders that affect both humans and animals, which are characterized by presences of protease‐resistance disease‐associated prion protein (PrPSc) in brains. In the present study, we optimized the Western blot assay for PrPSc with a precipitation procedure of streptomycin sulphate. After incubated with suitable amount of streptomycin sulphate, the detective sensitivity for PrPSc was remarkably improved. The precipitation of PrPSc was obviously influenced by pH value in the solution. Employs of PrPSc stock sample into various mimic specimens, including normal hamster brain homogenate, human cerebrospinal fluid and urine, demonstrated that streptomycin precipitation markedly increased the detective sensitivity of PrPSc, regardless in low concentration or in large volume. In addition, the PrPSc from a human brain tissue of familiar Creutzfeldt–Jakob disease (fCJD) was efficiently precipitated with streptomycin sulphate. As a sensitive, specific, rapid and flexible protocol for PrPSc, the protocol in this study has the potential, alone or combined with other techniques, to detect low levels of PrPSc in the specimens not only from central nerve system, but also from peripheral organs or fluids.
An AerosolizedBrucellaspp. Challenge Model for Laboratory Animals
Authors: Olsen S. C. , Waters W. R. , Stoffregen W. S.
Published: September 2007

To characterize the optimal aerosol dosage ofBrucella abortusstrain 2308 (S2308) andB. melitensis(S16M) in a laboratory animal model of brucellosis, dosages of 103–1010 colony forming units (CFU) were nebulized to mice. Although tissue weights were minimally influenced, total CFU per tissues increased beginning at 106–107 CFU dosages, with 109 CFU appearing to be an optimal dosage for S16M or S2308 aerosol delivery. At 12 weeks after vaccination with 107 CFU ofB. abortusstrain RB51 (SRB51) or saline (control), mice were challenged intraperitoneally (i.p.) (6.4 × 104 CFU) or via aerosol (1.76 × 109 CFU) with S2308. Mice vaccinated with SRB51 had reduced (P < 0.05) splenic, liver and lung colonization (total CFU and CFU/g) after i.p. challenge with S2308 as compared with control mice after i.p. S2308 challenge. Control and SRB51‐vaccinated mice did not differ (P > 0.05) in splenic, liver or lung colonization after aerosol S2308 challenge. Failure to demonstrate vaccine protection was not because of a high aerosol challenge dosage as colonization of spleen and liver tissues was lower (P < 0.05) after aerosol challenge when compared with control mice after i.p. S2308 hallenge.
Salmonella enterica Infections in Spanish Swine Fattening Units
Authors: García‐Feliz C. , Collazos J. A. , Carvajal A. , Vidal A. B. , Aladueña A. , Ramiro R. , De La Fuente M. , Echeita M. A. , Rubio P.
Published: September 2007

The present study is the first conducted in Spain to estimate the bacteriological herd prevalence of Salmonella enterica in fattening units and to describe the Salmonella serovar diversity on these farms using a sample representative of the entire swine population. For this purpose, 10 faecal samples were collected from 10 different pens containing pigs close to market weight in a total of 232 fattening units. Total sample size was proportionally distributed according to the fattener census in each of the regions of the country and all the samples were examined by culture of 25 g of faecal material. One hundred (43.1%) farms had at least one Salmonella‐positive sample (95% CI: 37–49.1%). Salmonella enterica was detected in 290 (12.5%) pooled faecal floor samples (95% CI: 11.2–13.8%). The apparent herd prevalence of salmonellosis was similar among multi‐site, finishing and farrow to finish farms. Overall, 24 different serovars were identified, with S. Typhimurium, S. Rissen and S. Derby being the most common both at herd and sample level. Results of phage typing were available for the 91 isolates of S. Typhimurium. A total number of 10 different phage types were identified, with DT 193 being the most frequent. Phage types DT 104, DT 104b and DT U302, which have been associated with several multi‐resistant patterns, accounted for 23% and 29% of the Typhimurium total isolates or Typhimurium infected farms respectively.
Extraintestinal Pathogenic Escherichia coli‐Induced Acute Necrotizing Pneumonia in Cats
Authors: Sura R. , Van Kruiningen H. J. , DebRoy C. , Hinckley L. S. , Greenberg K. J. , Gordon Z. , French R. A.
Published: September 2007

Extraintestinal pathogenic Escherichia coli (ExPEC) are pathogens involved in several disease conditions, ranging from urinary tract infection to meningitis in humans and animals. They comprise epidemiologically and phylogenetically distinct strains, affecting most species and involving any organ or anatomical site. Here, we report fatal cases of necrotizing pneumonia in cats. Over a 1‐week period, 13 cats from an animal shelter in Stamford, Connecticut were presented for necropsy. All had a clinical history of acute respiratory disease. The gross and microscopic findings for all the cats were consistent. Escherichia coli was uniformly isolated from the lungs of all the tested cats. All the isolates were haemolytic, genetically related as determined by enterobacterial repetitive intergenic consensus PCR, and harboured genes encoding for cytotoxic necrotizing factor‐1 and fimbriae and adhesions that are characteristic of ExPEC, implying a point source clonal outbreak. As cats are common household pets, this report raises concerns regarding zoonotic potential (in either direction) for these ExPEC strains.
In Vitro Quantification of Guidewire Flow-Obstruction Effect in Model Coronary Stenoses for Interventional Diagnostic Procedure
Authors: Ashtekar Koustubh D. , Back Lloyd H. , Khoury Saeb F. , Banerjee Rupak K.
Published: September 2007

The objective is to quantify the guidewire (diameter of 0.35mm) flow-obstruction effect in the in vitro model coronary stenoses in relation to trans-stenotic pressure drop, Δp, fractional flow reserve (gFFR; “g” represents FFR measurement with guidewire insertion) and coronary flow reserve (gCFR) for steady and pulsatile physiological flows. The sensor tipped pressure or flow measuring guidewire insertion through stenotic lumen increases the trans-stenotic pressure drop or reduces the pharmacologically induced hyperemic flow in the coronary arteries with plaques. These hemodynamic changes may cause error in true FFR and CFR measurements, especially for intermediate coronary stenosis. To quantify guidewire flow-obstruction effect, simultaneous measurements of trans-stenotic pressures and flow were performed by two methods: (a) guidewire based measurements (gCFR and gFFR by inserting sensor tipped guidewire) and (b) true physiological measurements (CFR by in-line Doppler flow cuff and FFR by the radially drilled pressure ports in three epicardial coronary stenotic test sections, postangioplasty, intermediate, and preangioplasty). The diagnostic parameters measured before guidewire insertion (CFR and FFR) and during guidewire insertion (gCFR and gFFR) were validated numerically and correlated with the new diagnostic parameter “lesion flow coefficient (LFC).” There was significant flow reduction with increased trans-stenotic pressure drop due to guidewire insertion. The FFR-gFFR and CFR-gCFR correlations were FFR=0.92×gFFR+0.097(R2=0.99) and CFR=0.91×gCFR+0.44(R2=0.99), respectively, where gCFR is reported from clinical pressure-flow data. Similar highly regressed (R2>0.9) correlations were obtained for LFC and gLFC with flow ratios and pressure ratios. There was significant difference between steady and pulsatile pressure drops for the same mean flow with and without guidewire insertion. The trans-stenotic hemodynamics was altered due to guidewire insertion. The true FFR and CFR were underestimated because of guidewire insertion. Hence, the FFR-gFFR and CFR-gCFR correlations can be used to find out true FFR and CFR from clinically measured values (i.e., gFFR and gCFR). In addition, the gLFC-gCFR and gLFC-gFFR were correlated significantly for post- and preangioplasty conditions.
Miniaturized Cutting Tool With Triaxial Force Sensing Capabilities for Minimally Invasive Surgery
Authors: Valdastri Pietro , Houston Keith , Menciassi Arianna , Dario Paolo , Sieber Arne , Yanagihara Masaru , Fujie Masakatsu
Published: September 2007

This paper reports a miniaturized triaxial force sensorized cutting tool for minimally invasive robotic surgery. This device exploits a silicon-based microelectromechanical system triaxial force sensor that acts as the core component of the system. The outer diameter of the proposed device is less than 3mm, thus enabling the insertion through a 9 French catheter guide. Characterization tests are performed for both normal and tangential loadings. A linear transformation relating the sensor output to the external applied force is introduced in order to have a triaxial force output in real time. Normal force resolution is 8.2bits over a force range between 0N and 30N, while tangential resolution is 7 bits over a range of 5N. Force signals with frequencies up to 250Hz can successfully be detected, enabling haptic feedback and tissue mechanical properties investigation. Preliminary ex vivo muscular tissue cutting experiments are introduced and discussed in order to evaluate the device overall performances.
Design of a Calibration Phantom for Measuring the Temporal Resolution of a Tomographic Imaging Device
Authors: Slocum Alexander H. , Jones Stephen E. , Gupta Rajiv
Published: September 2007

This paper describes the design and development of a calibration phantom to be used to aid in the calculation of the temporal resolution of tomographic imaging devices. Current practice for characterizing the dynamic response of a tomographic imaging device, such as a computed tomography or magnetic resonance imaging machine, uses image acquisition time as a surrogate for temporal resolution. At present, no standard method for describing the temporal resolution of a tomographic imaging device exists. Similar to the spatial modulation transfer function (MTF) used for characterizing spatial resolution, the concept of temporal MTF (t-MTF) can be used to enable characterization of temporal resolution. A scanner’s t-MTF represents the percentage amplitude modulation transfer in the image as a function of the input frequency. The calibration phantom uses slotted disks, each mounted to the rotating ring gear of a planetary gear assembly. The sun gears of each planetary gear set are driven from a common shaft to create differential speed sectors, allowing for about two decades of input frequencies to be obtained using a single motor and driveshaft. Preliminary results show a monotonic decline in the modulation transfer as the input frequency is increased. As expected, there is more modulation transfer at lower frequency and less modulation transfer at high frequency. Analogous to the spatial resolution, one can define the frequency for which there is 10% modulation transfer as the temporal resolution of a scanner.
Foxp3 Is Required Throughout the Life of a Regulatory T Cell
Authors: Lopes Jared E. , Soper David M. , Ziegler Steven F.
Published: September 2007

The
forkhead
family transcription factor Foxp3 is critical for the development and function of CD4
+
CD25
+
regulatory T cells (Tregs). A series of reports have begun to shed light on the precise role of Foxp3 in the regulation of the Treg transcriptome. Foxp3 can bind to specific gene elements, thereby altering transcription of target genes directly, and Foxp3 can alter the expression of genes encoding other transcription factors, thereby having an indirect effect on the transcription of target genes. Cells retaining aspects of Treg differentiation persist in the absence of Foxp3, which is suggestive of a Foxp3-independent aspect of Treg biology.
The Surprising Catch of a Voltage-Gated Potassium Channel in a Neuronal SNARE
Authors: Mohapatra Durga P. , Vacher Helene , Trimmer James S.
Published: September 2007

Among ion channels, voltage-gated calcium channels have been considered unique in their ability to mediate signaling events independent of the flow of ions through their pore. A voltage-gated potassium channel termed Kv2.1 has been identified as playing a role remarkably similar to one ion-independent function of calcium channels, facilitating regulated exocytosis through a direct interaction with a t-SNARE [soluble NSF (
N
-ethylmaleimide–sensitive factor) attachment protein receptor] component of the vesicle release machinery. Kv2.1 overexpression enhances depolarization-induced secretion from the neuroendocrine-like PC12 cell line, and a nonconducting Kv2.1 mutant can accomplish the same feat. Kv2.1 interacts directly with syntaxin 1A, a plasma membrane t-SNARE component of the vesicle docking and fusion apparatus. Deletion of the syntaxin 1A–binding segment from Kv2.1 abolishes its ability to promote vesicle release, supporting a mechanism whereby Kv2.1 presumably transfers voltage-dependent conformational changes induced by membrane depolarization to interacting t-SNAREs to affect exocytosis. Kv2.1, a major mediator of electrical events in central neurons, cardiac and smooth muscle, and pancreatic β cells, must now also be recognized as a physical mediator of secretion. That Kv2.1 is phosphorylated at numerous sites within the syntaxin 1A binding segment raises the possibility that its role in secretion may be dynamically regulated by diverse signaling events.
FOXP3
Is an X-Linked Tumor Suppressor
Authors: Adler Elizabeth M.
Published: September 2007

Many breast cancers occur in conjunction with overexpression of the
ERBB2
oncogene (also known as
HER-2
or
Neu
). Although gene amplification can lead to
ERBB2
overexpression, some human cancers overexpress
ERBB2
even without gene amplification. Zuo
et al
. noticed that a large percentage of older female mice heterozygous for a mutation in the
Foxp3
gene (

Foxp3
sf/+

) developed spontaneous cancers; about 60% of these were mammary carcinomas. Further, these mice showed increased susceptibility to developing mammary cancer in response to treatment with the carcinogen DMBA together with progesterone. Analysis of normal mammary epithelial tissue and mammary cancer cells microdissected from heterozygous mice revealed that the wild-type allele of
Foxp3
was expressed in the former and silenced in the latter, which also showed increased
ErbB2
expression. Chromatin immunoprecipitation analysis indicated that a Foxp3 fusion protein, which decreased
ErbB2
expression when transfected into a mouse mammary tumor cell line (TSA), bound the
ErbB2
promoter, and mutational analysis of reporter activity combined with electrophoretic mobility shift assay indicated that Fox3p binding was required for repression of
ErbB2
.
FOXP3
silencing with siRNA in normal human mammary epithelial cells led to an increase in
ERBB2
expression, whereas induction of
FOXP3
expression in a breast cancer cell line with
ERBB2
gene amplification decreased ErbB2 abundance at the cell surface. Defects in
FOXP3
were observed in human breast cancer cell lines and breast cancer tissues, and the presence of such defects showed a correlation with increased abundance of ErbB2. Intriguingly, transfection of
Foxp3
into breast cancer cell lines reduced their in vitro and in vivo growth. Thus, the authors conclude that
FOXP3
is a tumor suppressor that regulates
ERBB2
. Medema and Burgering discuss the implications of X chromosome inactivation with regard to X-linked tumor suppressors and the possibility that the role of Foxp3 in development of regulatory T cells may contribute to its tumor suppressor activity.

T. Zuo, L. Wang, C. Morrison, X. Chang, H. Zhang, W. Li, Y. Liu, Y. Wang, X. Liu, M. W. Y. Chan, J.-Q. Liu, R. Love, C.-g. Liu, V. Godfrey, R. Shen, T. H.-M. Huang, T. Yang, B. K. Park, C.-Y. Wang, P. Zheng, Y. Liu,
FOXP3
is an X-linked breast cancer suppressor gene and an important repressor of the
HER-2/ErbB2
oncogene.
Cell
129
, 1275-1286 (2007).
[PubMed]

R. H. Medema, B. M. Th. Burgering, The X factor: Skewing X inactivation towards cancer.
Cell
129
, 1253-1254 (2007).
[PubMed]
Calcium Channels Get ROCKed
Authors: Foley John F.
Published: September 2007

Three distinct α
1
subunit genes encode T-type Ca
2+
channels (Ca
V
3.1, Ca
V
3.2, and Ca
V
3.3), and these have nonredundant roles in pain transmission, cell proliferation, and cardiovascular function. Lysophosphatidic acid (LPA), a phospholipid involved in the development and growth of neurons, binds to members of a family of G protein-coupled receptors (GPCRs) called LPA1-5, of which LPA1 is the most abundant in the brain. LPA1 activates members of the Gα
i
, Gα
q
, and Gα
12/13
family of G proteins. Stimulated Gα
12/13
activates the Rho guanosine triphosphatase family member RhoA, which activates Rho-associated kinase (ROCK). ROCK isoforms phosphorylate (among other substrates) sodium and potassium channels. Previous studies have shown overlapping physiological responses to activation of either ROCK or T-type Ca
2+
channels, so Iftinca
et al
. investigated a role for ROCK in regulating T-type Ca
2+
channels. Whole-cell patch clamp assays on tsA-201 cells (a subclone of HEK 293 cells) transiently expressing rat Ca
V
3.1 showed a dose-dependent decrease in channel current in response to LPA. Experiments with pertussis toxin (to block Gα
i
) and protein kinase C inhibitors (to block signals downstream of Gα
q
) suggested that the Gα
12/13
pathway mediated the inhibitory effect of LPA on Ca
V
3.1 activity. Coexpression of dominant-negative mutants of Gα
12
and Gα
13
, or treatment with specific inhibitors of Rho or ROCK, substantially decreased the inhibitory effects of LPA. Mutation of Ser and Thr residues in putative ROCK-phosphorylation consensus sites (found between domains II and III) of Ca
V
3.1 resulted in mutant channels that did not respond to LPA. These consensus sites are conserved in all T-type Ca
2+
channels, and the authors found that whereas LPA treatment also inhibited the activity of Ca
V
3.3, it increased the activity of Ca
V
3.2. The authors performed experiments in neurons isolated from the lateral habenula (LHb) nucleus (predominantly expressing Ca
V
3.1) and the dorsal root ganglia (DRG, in which Ca
V
3.2 is the major channel) of rats and found that the effects of LPA (decreasing currents in LHb neurons and increasing currents in the DRG) were all blocked by inhibition of ROCK. Together, these data describe a new mechanism of regulation of the activity of T-type Ca
2+
channels and, given that T-type Ca
2+
channels and ROCK are coexpressed in other cell types, suggest that this regulation might be important for various physiological processes.

M. Iftinca, J. Hamid, L. Chen, D. Varela, R. Tadayonnejad, C. Altier, R. W. Turner, G. W. Zamponi, Regulation of T-type calcium channels by Rho-associated kinase.
Nat. Neurosci.
10
, 854-860 (2007).
[PubMed]
Decision-Making in the Fly
Authors: Stern Peter R.
Published: September 2007

Even the humble fruit fly has choices to make and uses a rudimentary brainlike organ known as the mushroom body in the process. Zhang
et al
. studied choice in
Drosophila
while the animals were confronted with competing visual cues. Decision-making was based on two processes, one linear and the other nonlinear. The switch from linear to nonlinear decision-making involved dopamine function in the mushroom body. Without the integrity of dopamine-mushroom body circuits, flies could make simple perceptual decisions based on the subtraction of the saliency of the conflict cues (color versus position) but lost the ability to amplify the discrepancy at some crucial points.

K. Zhang, J. Guo, Y. Peng, W. Xi, A. Guo, Dopamine-mushroom body circuit regulates saliency-based decision-making in
Drosophila
.
Science
316
, 1901-1904 (2007).
[Abstract]
[Full Text]
Yeast In Synch
Authors: Kelner Katrina L.
Published: September 2007

In the laboratory, yeast is usually grown in the presence of high glucose, and lab yeast is adapted for those conditions. If strains more typical of wild yeast are maintained under conditions with few nutrients, they will show a synchronous cycle of alternating respiratory and glycolytic metabolism. Cell division is confined to the glycolytic (or reductive) phase. Chen
et al
. hypothesized that this occurs to prevent replication from taking place in an oxidizing environment, which could cause mutations. When cells were forced to divide during the oxidizing phase of the metabolic cycle by introducing one of several cell cycle mutations or treating with H
2
O
2
, an increased level of DNA mutation was indeed observed. In addition, a mutation in a DNA checkpoint kinase that links the cell and circadian cycles in other fungi disrupts cell synchrony, suggesting parallels between the metabolic and circadian cycles.

Z. Chen, E. A. Odstrcil, B. P. Tu, S. L. McKnight, Restriction of DNA replication to the reductive phase of the metabolic cycle protects genome integrity.
Science
316
, 1916-1919 (2007).
[Abstract]
[Full Text]
Allosteric Activation of Toll-Like Receptor Activity
Authors: Ray L. Bryan
Published: September 2007

One of the most fundamental questions in signal transduction--precisely how receptors transmit information across the cell membrane--is not well understood. Latz
et al
. addressed this problem by analyzing signaling by a receptor of the innate immune system, Toll-like receptor 9 (TLR9), and obtained some unexpected results. TLR9 recognizes the presence of invading microbes by detecting microbial DNA, which is relatively enriched in unmethylated CpG motifs. Not all CpG-containing DNA fragments are stimulatory to the TLR9 receptor, and DNA with GGGG sequences can actually inhibit receptor signaling. However, the authors found that stimulatory, inhibitory, and control DNA fragments bound TLR4 with similar affinities. Thus, simple ligand binding appears not to elicit receptor activation. When the authors used circular dichroism spectroscopy to analyze conformational changes in a fusion protein of the TLR ectodomain with the Fc fragment of immunoglobulin G, they found that only stimulatory DNA induced a conformation change in the receptor fusion protein. Further evidence for a change in the proximity of the cytoplasmic signaling portion of the receptor upon binding of stimulatory DNA in living cells was obtained in experiments monitoring fluorescence resonance energy transfer (FRET). For these experiments, the authors tagged full-length TLR9 molecules on their C termini with appropriate donor and acceptor molecules. These studies also showed that TLR9 existed as a preformed dimer before the receptor bound DNA. The authors also measured behavior of the cytoplasmic signaling side of the receptor by creating two fusion proteins that each expressed only half of the green fluorescent protein. In cells expressing both proteins, fluorescence (attributable to folding of the two GFP fragments, thus indicating very close apposition of the two receptor proteins) was observed only in the presence of stimulatory CpG-containing DNA and only in the endosomes. Together, the results indicate that allosteric changes induced by binding of stimulatory DNA, rather than ligand-induced oligomerization, is the key to activation of TLR9. Thus, the authors note, it may be possible to more specifically select useful therapeutic agents by focusing on inhibition of the activating allosteric changes rather than inhibition of ligand binding. Such agents would be welcome because chronic overactivation of TLRs leads to autoimmune disorders that result from excessive inflammatory responses initiated by these receptors. Kim
et al
. discuss the results.

E. Latz, A. Verma, A. Visintin, M. Gong, C. M. Sirois, D. C. G. Klein, B. G. Monks, C. J. McKnight, M. S. Lamphier, W. P. Duprex, T. Espevik, D. T. Golenbock, Ligand-induced conformational changes allosterically activate Toll-like receptor 9.
Nat. Immunol.
8
, 772-779 (2007).
[PubMed]

Y.-M. Kim, M. M. Brinkmann, H. L. Ploegh, TLRs bent into shape.
Nat. Immunol.
8
, 675-677 (2007).
[PubMed]
Introduction to Enzyme Kinetics: Assay of β-Galactosidase
Authors: Tillotson Joanne Kivela
Published: September 2007

This tutorial describes a colorometric enzyme assay to determine simple enzyme kinetics. It is designed as an introduction for beginning laboratory students in a biology or biochemistry course, and requires no previous understanding of chemistry. It may be used as an assignment to prepare students for a laboratory exercise in enzyme kinetics Through this tutorial, students will gain an understanding of the procedures for measuring enzyme activity, using the specific example of an assay for the bacterial enzyme, β-galactosidase.
A Biochemical Assay for Acetylcholinesterase Activity in PC12 Cells
Authors: Schwartz Paul J. , Blundon Jay A. , Adler Elizabeth M.
Published: September 2007

This lab describes two biochemical assays: One for measuring acetylcholinesterase activity and one for measuring protein concentration. Students learn how to manipulate small-volume samples, use a standard spectrophotometer or a microplate reader spectrophotometer, construct a standard curve, and normalize data. The lab is intended to be used in conjunction with a cell culture lab in which PC12 cells are exposed to various agents that influence their phenotypic state.
Converting Attraction to Repulsion
Authors: Adler Elizabeth M.
Published: September 2007

During nervous system development, axonal growth cones respond to attractive and repulsive cues that help them navigate to their targets. Wen
et al
. discovered that, whereas embryonic
Xenopus
spinal axons in an in vitro culture system were attracted to a gradient of bone morphogenetic protein 7 (BMP7) presented 4 to 8 hours after being placed in culture, BMP7 repelled the same axons after they had been cultured overnight. The type II BMP receptor (BMPRII) mediated both attractive and repulsive responses, and overexpression of a shortened form of BMPRII [which lacks a region that mediates interactions with LIM kinase I (LIMKI) but is dispensable for activation of Smad signaling] abolished BMP7-induced turning. A 32-amino-acid peptide that competitively inhibits LIMKI abolished attractive turning to BMP7, as did a dominant-negative LIMKI mutant, whereas neither affected repulsive turning. In neurons cultured overnight that overexpressed a dominant-negative mutant of the phosphatase Slingshot (SSH), however, the response to BMP7 was converted from repulsion to attraction. A combination of live imaging of the actin cytoskeleton, immunofluorescence analysis of the phosphorylation of XAC [
Xenopus
actin-depolymerizing factor (ADF)/cofilin], and expression of wild-type and mutant forms of XAC revealed that LIMKI and SSH regulated growth cone turning through phosphorylation of XAC. BMP7 elicited an increase in growth cone calcium concentration in neurons cultured overnight but not in those cultured for 6 hours. Manipulations designed to block calcium signaling failed to affect attractive turning in 4- to 8-hour cultures, whereas in overnight cultures these treatments abolished the BMP-dependent decrease in XAC phosphorylation and converted repulsive to attractive turning, as did pharmacological inhibition of calcineurin. The transient receptor potential channel TRPC1 was more abundant in overnight cultures than in 4- to 8-hour cultures, and expression of a dominant-negative TRPC1 mutant abolished BMP7-dependent repulsion in overnight cultures, whereas overexpression of wild-type TRPC1 elicited BMP7-dependent repulsion in 4- to 8-hour cultures. Thus, opposing actions of LIMKI and SSH on XAC phosphorylation appear to regulate the actin cytoskeleton and thereby growth cone turning, with calcium influx through TRPC1 switching attraction to repulsion by shifting the balance toward SSH.

Z. Wen, L. Han, J. R. Bamburg, S. Shim, G.-l. Ming, J. Q. Zheng, BMP gradients steer nerve growth cones by a balancing act of LIM kinase and Slingshot phosphatase on ADF/cofilin.
J. Cell Biol.
178
, 107-119 (2007).
[Abstract]
[Full Text]
Numb Cells Keep Moving
Authors: Foley John F.
Published: September 2007

Integrins are heterodimeric transmembrane receptors that bind to components of the extracellular matrix and are important for both cellular adhesion and migration. Clustering of activated integrins on the substrate-facing surface of the leading edge of a cell results in the recruitment of various proteins, including actin stress fibers, to form a focal adhesion complex (FAC). Cells migrate, in part, through the coordinated assembly of focal adhesions at the leading edge of a cell and the disassembly of such complexes at the rear of the cell. Integrins are thought to move from the rear of the cell to the front through recycling endosomes, although the mechanisms that mediate internalization of integrins are not fully understood. Numb is a cargo-specific adaptor protein that binds to several endocytic proteins and is perhaps best known for its role in down-regulating the signaling of Notch, a transmembrane receptor that affects cellular proliferation and differentiation. Nishimura
et al
. previously demonstrated that Numb localizes to the tips of growing axons in cultures of hippocampal neurons and that it plays a role in the internalization of the adhesion molecule L1, so they investigated a role for Numb in mediating integrin endocytosis and cell migration. The authors used immunohistochemistry to demonstrate the colocalization of Numb with clathrin-coated structures in various endothelial and epithelial cell cultures. Analysis of green fluorescent protein (GFP) fusions of wild-type and mutant Numb proteins showed that the α-adaptin-binding motif was necessary for the interaction of Numb with clathrin-coated structures. In a monolayer wounding assay, Numb polarized toward the leading edge of migrating cells (just behind the lamellipodium). Numb localized around focal adhesions, and this association was inhibited by disruption of polymerized actin (F-actin). Immunostaining demonstrated the colocalization of Numb and β
1
-integrin at focal adhesions, and recombinant Numb protein coprecipitated β
1
- and β
3
-integrin but not other components of FACs. Depletion of Numb by siRNA reduced the number of β
1
-integrin-positive endosomes in migrating HeLa cells and also markedly reduced the number of HeLa cells that migrated toward integrin ligands compared with control siRNA-treated HeLa cells. Coimmunoprecipitation experiments revealed that Numb bound to the PAR (for partitioning defective) polarization complex, PAR-3. This complex localizes to the leading edge of polarized migrating cells. One component of this complex, atypical protein kinase C (aPKC) phosphorylated Numb in vitro and in HeLa cells and, as a consequence, Numb no longer bound to integrins. The authors propose a model in which Numb binds to free integrin molecules (rather than disrupting FACs) and recruits them to clathrin-coated structures to initiate integrin recycling, and that the localization and function of Numb are negatively regulated by aPKC.

T. Nishimura, K. Kaibuchi, Numb controls integrin endocytosis for directional cell migration with aPKC and PAR-3.
Dev. Cell
13
, 15-28 (2007).
[PubMed]
Balanced on One Foot
Authors: Gough Nancy R.
Published: September 2007

Cd1 proteins bind lipid antigens and present them to T cells to initiate immune responses. Cd1d presents glycolipids with specific α-linked sugars to the natural killer T cells (NKT) for recognition by their T cell receptor (TCR) complex. Borg
et al
. report the crystal structure of Cd1 bound to α-galactosylceramide (α-GalCer) with the NKT TCR. They find that the TCR interaction is unlike that of TCRs with the peptide-presenting major histocompatibility proteins (pMHCs). In the case of the pMHC-TCR interaction, the TCR α and β chains both contact the pMHC (see Moody), revealing two binding site "footprints" on the surface of the pMHC. The crystal structures reported by Borg
et al
. show that NKT TCR only binds to the Cd1d-α-GalCer by the α subunit, with the β subunit showing limited interaction and mostly hanging over the side of Cd1d. Moody describes the two structures as the TCR standing with both feet contacting pMHC, whereas NKT TCR appears as a pirouette balanced on one foot at the Cd1d surface. The structure also shows that the α-linked galactose lies flat, optimizing the interaction surface between Cd1d and the NKT TCR, which may provide a basis for the selective recognition of α-linked sugars. This study reinforces the notion that glycolipid-antigen recognition is achieved through molecular mechanisms different from those for peptide-antigen recognition and cautions against casting the Cd1 lipid-presenting proteins as MHC-like.

N. A. Borg, K. S. Wun, L. Kjer-Nielsen, M. C. J. Wilce, D. G. Pellicci, R. Koh, G. S. Besra, M. Bharadwaj, D. I. Godfrey, J. McCluskey, J. Rossjohn, CD1d-lipid-antigen recognition by the semi-invariant NKT T-cell receptor.
Nature
448
, 44-49 (2007).
[PubMed]

D. B. Moody, How a T cell sees sugar.
Nature
448
, 36-37 (2007).
[PubMed]
Possible Role for Deacetylation in DNA Damage Response
Authors: Ray L. Bryan
Published: September 2007

An intricate signaling system exists in cells to detect damaged DNA, delay progression through the cell cycle, and initiate repair of the damage. Yuan
et al
. provide evidence for a new layer of regulation to this process, showing that the NBS1 protein is subject to regulation by acetylation and deacetylation on lysine residues. NBS stands for Nijmegen breakage syndrome, the disease caused by mutations in the gene encoding NBS1. Affected individuals show a range of symptoms that include sensitivity to radiation and predisposition to lymphoid cancers. NBS1 functions in a protein complex known as MRN (MRE-11-RAD50-NBS1) that participates in sensing and repairing DNA damage. NBS1 was acetylated in cells transfected with various acetyltransferases, and acetylation was enhanced if cells were treated with inhibitors of class III histone deacetylases (HDACs). Acetylation of NBS1 by transfected acetylases was also enhanced if the HDAC SIRT1 was depleted with RNAi. Modification of NBS1 by phosphorylation is critical for sensing of DNA damage and for the cell cycle checkpoint mechanism that delays DNA replication until repair is completed. Overexpression of an acetylase and consequent acetylation of NBS1 decreased phosphorylation of NBS1. Transfected acetylase also inhibited the cell cycle checkpoint function that prevents DNA synthesis after exposure of cells to ionizing radiation. This effect was not observed, though, when cells expressed a mutant of NBS1 resistant to acetylation. In mouse embryo fibroblasts that did not express the SIRT1 deacetylase, in which NBS1 was expected to be highly acetylated and thus poorly phosphorylated, checkpoint function was also compromised. Thus, the authors propose that crosstalk between acetylation and phosphorylation mechanisms may regulate responses to DNA damage.

Z. Yuan, X. Zhang, N. Sengupta, W. S. Lane, E. Seto, SIRT1 regulates the function of the Nijmegen breakage syndrome protein.
Mol. Cell
27
, 149-162 (2007).
[PubMed]
Redox Receptor Regulation Revealed
Authors: Ray L. Bryan
Published: September 2007

Thioredoxin-1 (Trx1) is a thiol-disulfide oxidoreductase that can reduce transiently formed disulfide bonds in substrate proteins. The enzyme is released from lymphocytes and has extracellular cytokine-like actions, but the mechanisms by which it produces such effects are not known. Schwertassek
et al
. used a modified form of Trx1 in a substrate trapping assay (in which the engineered Trx1 formed long-lived mixed disulfide intermediates with its targets) to search for cell-surface proteins that might be modified by the enzyme. An affinity tag on the modified Trx1 allowed purification of the complexes and subsequent identification of associated proteins by mass spectrometry. In the human Epstein-Barr virus--transformed lymphoblastoid B cell line LCL-721.220--the authors found just one primary target of Trx-1, a member of the tumor necrosis factor receptor (TNFR) superfamily known as TNFRSF8 or CD30. The association of the proteins was lost under reducing conditions and, although all TNFR superfamily members characteristically have cysteine-rich domains, association with Trx1 was specific to CD30. Modification of CD30 appeared to cause a conformational change in the receptor because recognition of the receptor by antibodies was altered. This action of Trx1 required the presence of a source of reducing equivalents. Incubation of Hodgkin’s lymphoma HDLM-2 cells decreased binding of recombinant soluble CD30 ligand to its receptor at the cell surface and, in the YT granular lymphoma cell line, blocked increased expression of the gene encoding the interleukin-2 receptor alpha chain, which is normally activated by CD30 signaling. The authors thus propose that excreted Trx1 may alter receptor function and may, in this way, link oxidative stress to lymphocyte function.

U. Schwertassek, Y. Balmer, M. Gutscher, L. Weingarten, M. Preuss, J. Engelhard, M. Winkler, T. P. Dick, Selective redox regulation of cytokine receptor signaling by extracellular thioredoxin-1.
EMBO J.
26
, 3086-3097 (2007).
[PubMed]
Growth Hormone and Development
Authors: Purnell Beverly A.
Published: September 2007

During development, genes are often transcribed in a temporally and spatially regulated manner. The murine growth hormone gene is differentially expressed in the developing pituitary gland. Lunyak
et al
. now examine the region surrounding the growth hormone gene and show that a repeated DNA sequence (short interspersed nuclear element B2) in the growth hormone locus functions as an insulator to produce a boundary for chromatin domains and limit the action of regulatory factors such as enhancers and silencers.

V. V. Lunyak, G. G. Prefontaine, E. Núñez, T. Cramer, B.-G. Ju, K. A. Ohgi, K. Hutt, R. Roy, A. García-Díaz, X. Zhu, Y. Yung, L. Montoliu, C. K. Glass, M. G. Rosenfeld, Developmentally regulated activation of a SINE B2 repeat as a domain boundary in organogenesis.
Science
317
, 248-251 (2007).
[Abstract]
[Full Text]
Cohesin Does the Business
Authors: Riddihough Guy
Published: September 2007

To ensure the sorting of a complete complement of chromosomes to both daughter cells in cell division, sister chromatids are bound together by a ring-shaped molecular complex called cohesin. The accurate repair of double-stranded lesions in DNA also relies on cohesion between homologous regions of sister chromatids. Both these processes are often misregulated in cancer. Cohesion has been thought to require ongoing DNA replication (see the Perspective by Watrin and Peters). Ünal
et al.
and Ström
et al
. now show in yeast that double-stranded breaks can induce cohesion in the absence of replication and that the deposition of cohesin is not limited to the region of the break but extends across the entire genome and, thus, may play a role in maintaining genome stability.

E. Watrin, J.-M. Peters, How and when the genome sticks together.
Science
317
, 209-210 (2007).
[Summary]
[Full Text]

E. Ünal, J. M. Heidinger-Pauli, D. Koshland, DNA double-strand breaks trigger genome-wide sister-chromatid cohesion through Eco1 (Ctf7).
Science
317
, 245-248 (2007).
[Abstract]
[Full Text]

L. Ström, C. Karlsson, H. B. Lindroos, S. Wedahl, Y. Katou, K. Shirahige, C. Sjögren, Postreplicative formation of cohesion is required for repair and induced by a single DNA break.
Science
317
, 242-245 (2007).
[Abstract]
[Full Text]
Keeping the (Kinase) Party Going: SLP-76 and ITK Dance to the Beat
Authors: Qi Qian , August Avery
Published: September 2007

The Tec-family protein tyrosine kinase IL-2–inducible T cell kinase (ITK) mediates T cell activation, as does the adaptor protein SLP-76 (SH2-domain–containing leukocyte protein of 76 kD), which forms a complex with ITK and other intracellular signaling enzymes. One of these enzymes is phospholipase C–γ1 (PLC-γ1), which mediates T cell receptor (TCR)–stimulated intracellular calcium mobilization leading to the activation of transcription factors such as nuclear factor of activated T cells. The Src-family tyrosine kinase Lck and the Syk-family tyrosine kinase ζ chain–associated protein kinase of 70 kD (ZAP-70), together with ITK, are necessary for the phosphorylation of PLC-γ1 in response to TCR stimulation. ITK is thought to phosphorylate a specific tyrosine residue of PLC-γ1 that is required for its activation. The mechanism of activation of ITK appears to involve the interaction between SLP-76 and ITK, which not only initiates ITK activity but is also important to maintain the kinase activity of ITK. This suggests that SLP-76 acts as more than a neutral adaptor in mediating T cell activation; SLP-76 also directly influences the kinase activity of ITK, allowing ITK to phosphorylate PLC-γ1.
Oncogenic Ras-Induced Interleukin-6 Promotes Tumor Growth
Authors: Foley John F.
Published: September 2007

Ras is a membrane-bound small guanosine triphosphatase (GTPase) that mediates signals from receptor tyrosine kinases to members of the mitogen-activated protein kinase (MAPK) family. Mutations in Ras that render it constitutively active are oncogenic, leading to the uncontrolled proliferation of cells and the growth of tumors. Many tumors are associated with such mutants of Ras (such as Ras
G12V
) or with mutations in either upstream activators of Ras or its downstream effectors. However, Ras has not proved to be a very successful pharmacological target, so Ancrile
et al
. turned their attention to secreted factors associated with Ras-induced tumors. One such factor is the cytokine interleukin-6 (IL-6). IL-6 has both pro- and anti-inflammatory properties, and its abundance is increased in the sera of patients with pancreatic cancer, which is associated with oncogenic Ras mutations. The authors used enzyme-linked immunosorbent assays (ELISAs) to measure IL-6 secretion from a transformed human kidney cell line that expressed 4-hydroxytamoxifen (4-OHT)-inducible Ras
G12V
. The abundance of IL-6 protein in 4-OHT-treated cultures was much higher than in noninduced cultures, and these results were confirmed at the level of
IL-6
gene transcription by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assays. When Ras
G12V
-expressing cells were placed under the skin of the flanks of mice, tumors developed. However, tumors that developed from cells treated with IL-6-specific short hairpin RNAs (shRNAs) were much smaller than those that developed from control shRNA-treated cells. Topical application of the carcinogen 7,12-dimethylbenzanthracene (DMBA) followed by 12-
O
-tetradecanoylphorbol-13-acetate (TPA) produces papillomas with a high incidence of oncogenic Ras mutations. The authors performed DMBA and TPA treatments of both wild-type and
IL-6
–/–
mice and found that
IL-6
–/–
mice had fewer and smaller tumors than did treated wild-type mice and also developed these tumors later than did their wild-type counterparts. Flow cytometry revealed that Ras
G12V
-induced tumor cells did not express IL-6 receptors, indicating that these cells did not need IL-6 for proliferation. However, immunohistochemical analysis revealed that tumors that developed from IL-6-specific shRNA-treated Ras
G12V
-expressing cells contained far fewer endothelial cells than did tumors that developed from control shRNA-treated Ras
G12V
-expressing cells, suggesting a role for Ras
G12V
-induced IL-6 in stimulating angiogenesis, leading to tumor growth. Finally, using the same in vivo tumor model, the authors showed that treatment of mice with a neutralizing antibody against IL-6 led to smaller tumors (and a slower growth rate) than were observed in control antibody-treated mice. These data suggest that targeting IL-6 in Ras-related cancers might be of therapeutic benefit.

B. Ancrile, K.-H. Lim, C. M. Counter, Oncogenic Ras-induced secretion of IL6 is required for tumorigenesis.
Genes Dev
.
21
, 1714-1719 (2007).
[Abstract]
[Full Text]