bluuebunny/crossref_metadata_2025 huggingface dataset:
DANGER, New Regulator of IP
3
R Calcium-Release Channel
Authors: Ray L. Bryan
Published: December 2006

The inositol 1,4,5-trisphosphate receptor (IP
3
R) functions as a sensor for the second messenger IP3 and as a channel protein that allows release of Ca
2+
from intracellular stores in response to activated receptors that are linked to production of IP
3
. However, there are some 2000 extra amino acids in the large IP
3
R protein in between the region that confers IP
3
binding and the Ca
2+
channel itself, and a number of proteins have been identified that interact with the IP
3
R in this region to regulate its function. Van Rossum
et al
. used a yeast 2-hybrid screen to search for other IP
3
R-interacting proteins in mammalian cDNA libraries and identified a new one they call DANGER. The authors confirmed interaction of the endogenous proteins by immunoprecipitation from cultured PC12 and showed colocalization with IP
3
Rs near the plasma membrane by immunofluorescence microscopy. Addition of glutathione
S
-transferase­-tagged DANGER to microsomal fractions from COS7 cells expressing transfected IP
3
R enhanced the sensitivity of the channel to inhibitory feedback by Ca
2+
. Normally, supraphysiological concentrations of Ca
2+
are needed to decrease IP
3
-dependent release of Ca
2+
through the receptor, but when DANGER was present, moderate concentrations of Ca
2+
(0.8 to 3.8 μM) almost abolished Ca
2+
release. Single-channel recordings of IP
3
R activity showed that DANGER reduced the channel open probability by decreasing the opening rate of the channel. The authors propose that DANGER appears to cause allosteric regulation of the IP
3
R that enhances the Ca
2+
sensitivity of the receptor such that Ca
2+
becomes a major regulator of the IP
3
R in cells expressing DANGER.

D. B. van Rossum, R. L. Patterson, K.-H. Cheung, R. K. Barrow, V. Syrovatkina, G. S. Gessell, S. G. Burkholder, D. N. Watkins, J. K. Foskett, S. H. Snyder, DANGER, a novel regulatory protein of inositol 1,4,5-trisphosphate-receptor activity.
J. Biol. Chem.
281
, 37111-37116 (2006).
[Abstract]
[Full Text]
Startled by a Lack of Zn
2+
(Modulation)
Authors: Gough Nancy R.
Published: December 2006

Zinc is present in synaptic vesicles and can be detected in synaptic regions. Various proteins that function in synaptic transmission (neurotransmitter receptors and transporters) are modulated by zinc, including glycine receptors (GlyRs), which are potentiated at low concentrations of zinc and inhibited by high concentrations of zinc. On the basis of previous experiments in which residues critical for zinc modulation had been determined, Hirzel
et al
. created knockin mice containing a mutation in the gene encoding the GlyR α1 subunit [
Glra1
(D80A)] in which zinc was no longer able to potentiate GlyR function. The mutant mice showed a neuromotor phenotype very similar to a human disorder called hyperekplexia; the mouse version includes increased acoustic startle reflex, altered visual signaling, inducible tremor, and altered gait. Spinal neurons cultured from wild-type and mutant mice showed that glycine responsiveness in the absence of added Zn
2+
was unchanged. However, neurons from the mutant mice showed decreased potentiation of glycine-mediated currents in the presence of 5 μM Zn
2+
without any impairment of inhibition of glycine-mediated currents in the presence of 100 μM Zn
2+
. In spinal cord cultures, application of 5 μM Zn
2+
increased the amplitude of miniature inhibitory postsynaptic currents (mIPSCs) in the wild-type cultures but decreased the amplitude of the mIPSCs in the mutant cultures. Slice preparations containing hypoglossal motoneurons did not exhibit altered IPSCs in response to exogenous Zn
2+
, suggesting that the slices contained sufficient free Zn
2+
to maximally potentiate GlyR-mediated currents. However, if free Zn
2+
was chelated, the amplitude of the IPSCs decreased in the wild-type slices and addition of 5 μM Zn
2+
restored the IPSC amplitude. Chelation of Zn
2+
had no effect on slices from the mutant mice. The role of zinc in synaptic signaling has been controversial, but even the skeptics should find that these results provide compelling evidence for an important neuromodulatory role for zinc in inhibitory synaptic transmission (see Kay
et al
.). Readers are invited to discuss this article and the general topic of zinc in cell signaling in the open forum "Questions and Controversies in Zinc Signaling" (see Related Resources).

K. Hirzel, U. Müller, A. T. Latal, S. Hülsmann, J. Grudzinska, M. W. Seeliger, H. Betz, B. Laube, Hyperekplexia phenotype of glycine receptor α1 subunit mutant mice identifies Zn
2+
as an essential endogenous modulator of glycinergic neurotransmission.
Neuron
52
, 679-690 (2006).
[PubMed]

A. R. Kay, J. Neyton, P. Paoletti, A startling role for synaptic zinc.
Neuron
52
, 572-574 (2006).
[PubMed]
Turn on the PIPs
Authors: Stern Peter
Published: December 2006

Many cellular proteins are inhibited when phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P
2
] is hydrolyzed by phospholipase C and reactivated when a kinase restores the PtdIns(4,5)P
2
(see the Perspective by McLaughlin, published online 9 November). Whether depletion of PtdIns(4,5)P
2
is the direct signal closing KCNQ potassium channels, an important ion channel in the nervous system, upon receptor activation is still controversial. Suh
et al
. (published online 21 September) now show that a loss of PtdIns(4,5)P
2
indeed suffices to close the channel. Furthermore, KCNQ channel opening was stimulated by synthesis of extra PtdIns(4,5)P
2
but only minimally affected by production of extra PtdIns(3,4,5)P
3
. Small GTPases (guanosine triphosphatases) have many roles in cellular regulation and often transiently associate with the plasma membrane. Heo
et al
. (published online 9 November) monitored the cellular localization of fluorescently tagged small GTPases and found that the majority of the enzymes that associated with the membrane contained a cluster of positively charged amino acids. Suspecting that these regions might bind negatively charged lipids, the authors showed that PtdIns(4,5)P
2
and PtdIns(3,4,5)P
3
were required for targeting the GTPases to the plasma membrane. The lipids may act as signaling hubs that control multiple aspects of cellular control systems.

S. McLaughlin, Tools to tamper with phosphoinositides.
Science
314
, 1402-1403 (2006).
[Summary]
[Full Text]

B.-C. Suh, T. Inoue, T. Meyer, B. Hille, Rapid chemically induced changes of PtdIns(4,5)P
2
gate KCNQ ion channels.
Science
314
, 1454-1457 (2006).
[Abstract]
[Full Text]

W. D. Heo, T. Inoue, W. S. Park, M. L. Kim, B. O. Park, T. J. Wandless, T. Meyer, PI(3,4,5)P
3
and PI(4,5)P
2
lipids target proteins with polybasic clusters to the plasma membrane.
Science
314
, 1458-1461 (2006).
[Abstract]
[Full Text]
Progesterone and Breast Cancer
Authors: Kiberstis Paula A.
Published: December 2006

Mutations in the breast cancer susceptibility gene
BRCA1
greatly increase a woman’s risk of developing breast and ovarian cancers. Why do these mutations predominantly affect hormone-responsive tissues when the mutant gene is widely expressed throughout the body? Poole
et al
. (see the news story by Marx) suggest that this tissue specificity is caused in part by BRCA1-mediated effects on signaling by the hormone progesterone. Mammary epithelial cells (MECs) of
Brca-1/p53
-deficient mice accumulated high levels of progesterone receptors, probably through defective degradation by the proteasome, and developed aberrant proliferation of the MECs. Treatment with the progesterone antagonist mifepristone (RU 486) prevented or delayed mammary tumor development in the mice.

A. J. Poole, Y. Li, Y. Kim, S.-C. J. Lin, W.-H. Lee, E. Y.-H. P. Lee, Prevention of
Brca1
-mediated mammary tumorigenesis in mice by a progesterone antagonist.
Science
314
, 1467-1470 (2006).
[Abstract]
[Full Text]

J. Marx, Squelching progesterone’s signal may prevent breast cancer.
Science
314
, 1370 (2006).
[Summary]
[Full Text]
Of Genes and Gut Reactions
Authors: Kiberstis Paula A.
Published: December 2006

Inflammatory bowel diseases (IBDs) such as Crohn’s disease and ulcerative colitis are thought to be caused by an inappropriate immune response to commensal intestinal bacteria. There is strong evidence that these disorders have a genetic component; for example, individuals carrying specific sequence variants of the
NOD2/CARD15
gene are at increased risk. Now, in a genome-wide association study, Duerr
et al
. (published online 26 October; see the Perspective by Cardon) find that a rare sequence variant of the gene encoding the receptor for interleukin-23 (
IL23R
) significantly lowers an individual’s risk of developing IBDs. Interleukin-23 is a cytokine that has attracted increasing attention because of its role in a wide range of chronic inflammatory diseases in mouse models, including IBDs, multiple sclerosis, and arthritis.

R. H. Duerr, K. D. Taylor, S. R. Brant, J. D. Rioux, M. S. Silverberg, M. J. Daly, A. H. Steinhart, C. Abraham, M. Regueiro, A. Griffiths, T. Dassopoulos, A. Bitton, H. Yang, S. Targan, L. W. Datta, E. O. Kistner, L. P. Schumm, A. T. Lee, P. K. Gregersen, M. M. Barmada, J. I. Rotter, D. L. Nicolae, J. H. Cho, A genome-wide association study identifies
IL23R
as an inflammatory bowel disease gene.
Science
314
, 1461-1463 (2006).
[Abstract]
[Full Text]

L. R. Cardon, Delivering new disease genes.
Science
314
, 1403-1405 (2006).
[Summary]
[Full Text]
Turing Patterning in the Mouse Hairs
Authors: Purnell Beverly
Published: December 2006

More than 50 years ago, Alan Turing provided a theoretical explanation of biological pattern formation through a hypothesis of reaction-diffusion, whereby patterns, such as that for hair follicles or feather distribution, can form as a result of positive and negative feedback regulation of an inhibitor and activator. Turing models have since been used to account for patterns in many chemical systems but have not been successful in explaining biological patterning in developmental model systems such as the fly. Sick
et al
. have now examined hair follicle arrangements in mice that arise through the WNT activator protein and its inhibitor DKK and show through computation modeling that reaction-diffusion can account for the patterning observed.

S. Sick, S. Reinker, J. Timmer, T. Schlake, WNT and DKK determine hair follicle spacing through a reaction-diffusion mechanism.
Science
314
, 1447-1450 (2006).
[Abstract]
[Full Text]

P. K. Maini, R. E. Baker, C.-M. Chuong, The Turing model comes of molecular age.
Science
314
, 1397-1398 (2006).
[Summary]
[Full Text]
What Is So Special About
Oskar
Wild?
Authors: O’Gorman William , Akoulitchev Alexandre
Published: December 2006

The amazing world of regulatory noncoding RNA has been at the center of biologists’ attention in many different fields, from structural biology to transcriptional regulation and cell signaling. The latest example comes from developmental biology. A mutation in the
Drosophila
gene
Oskar
reveals a novel developmental function for the 3′ untranslated region (UTR) of the
oscar
mRNA. This study further suggests that, when transcribed, the noncoding parts of the genome may well carry essential regulatory functions fundamental for the coordinated gene expression and development of multicellular organisms.
PI3 Kinases in Cancer: From Oncogene Artifact to Leading Cancer Target
Authors: Zhao Jean J. , Roberts Thomas M.
Published: December 2006

At the time of its discovery in the 1980s, the lipid kinase activity found associated with certain viral oncoproteins, which we now know to originate from the class IA phosphatidylinositide 3-kinases (PI3Ks), was thought by many to be a purification artifact. Subsequent work identified these enzymes as key regulators of cell signaling pathways that control various cellular processes including proliferation, motility, survival, and apoptosis. It is now clear that the PI3K pathway is activated by genetic or epigenetic alterations in a large proportion of human tumors, and a search for small molecule inhibitors of PI3K activity is currently a major effort of the pharmaceutical industry. However, because of the importance of PI3Ks in normal physiology, the road to therapeutics might not be smooth and could require further dissection of PI3K signaling pathways. In particular, it may be important to distinguish among the roles of the various isoforms of class 1A PI3K in both normal physiology and tumorigenesis.
Translation Initiator Drags Mad Protein to Destruction
Authors: Ray L. Bryan
Published: December 2006

Eukaryotic translation initiation factor 4a (eIF
4
A) got its name by functioning to initiate translation, but new evidence shows it to have an independent role influencing the abundance of two
Drosophila
signaling molecules, the SMAD proteins Mad and Medea. Li and Li were led to this surprising conclusion when exploring an earlier finding that a dominant-negative mutant of eIF
4
A suppressed haploinsufficiency of the gene encoding Decapentaplegic (Dpp), the fly ortholog of BMP (bone morphogenetic protein--a member of the transforming growth factor-β superfamily). Further genetic analysis showed that reduced amounts of eIF
4
A helped compensate for mutations that reduced Dpp signaling in embryos and adult flies. Flies expressing the dominant-negative eIF
4
A had increased amounts of Mad protein, suggesting that the loss of eIF
4
A function was decreasing the degradation of Mad. In keeping with this idea, overexpression of eIF
4
A inhibited Dpp signaling and decreased the abundance of Mad. The authors used various coimmunoprecipitation experiments, including analysis of the endogenous proteins in embryo extracts, to show interaction of eIF
4
A with Mad. Experiments with a dominant-negative form of eIF
4
A that inhibits translation indicated that the effects of eIF
4
A on Dpp signaling are independent of its role in translation. Rather, genetic experiments suggested that eIF
4
A could act synergistically with the ubiquitin E3 ligase DSmurf, which regulates the abundance of Mad by promoting its degradation. The synergy suggests that eIF
4
A influenced the same process, but other experiments showed that eIF
4
A could also influence Mad abundance independently of DSmurf. The authors speculate that eIF
4
A may function as an adaptor to link SMAD proteins to the protein degradation machinery. Affolter and Pyrowolakis provide commentary.

J. Li, W. X. Li, A novel function of
Drosophila
eIF4A as a negative regulator of Dpp/BMP signalling that mediates SMAD degradation.
Nat. Cell Biol.
8
, 1407-1414 (2006).
[PubMed]

M. Affolter, G. Pyrowolakis, eIF4A goes beyond translation.
Nat. Cell Biol.
8
, 1319-1321 (2006).
[PubMed]
Serine Acylation of Wnt
Authors: Gough Nancy R.
Published: December 2006

Takada
et al
. provide evidence for a new type of protein acylation. Using transfected mouse L cells expressing Wnt-3a or Wnt-5a, the authors show that the proteins are acylated by incorporation of radiolabeled palmitic acid. This was not surprising, because Wnt had previously been reported to be palmitoylated on Cys
77
. What was surprising was that the acylation of Wnt-3a was not diminished by mutation of the Cys to alanine. The acylation site in Wnt-3a was mapped to Ser
209
using deletion and point mutation constructs. Mass spectrometry analysis showed that Ser
209
was modified by the addition of the monounsaturated lipid palmitoleic acid (C16:1). Porcupine is a putative O-acyltransferase, and in
Drosophila
porcupine is required for Wnt (called Wingless) secretion. Reduction of porcupine by small interfering RNA decreased the secretion of Wnt-3a and promoted the retention of the endoplasmic reticulum. Furthermore, the S209A mutant of Wnt-3a was also not secreted from the cells and was retained in the endoplasmic reticulum (ER). In the system used, Wnt-3a was predominantly in the ER because of the overexpression of the protein; therefore, to assess the effects of Ser
209
acylation of transit through the biosynthetic pathway, the authors treated the cells with cycloheximide to block new protein synthesis. The functional consequences of acylation at Ser
209
were assessed in a
Xenopus
development assay. Injection of embryos with Wnt-3a RNA causes ectopic axis formation; however, injection of the S209A mutant RNA did not cause any notable phenotype. These results open a new window into protein acylation with the addition of unsaturated lipids as modifiers of proteins (see Hausmann and Basler).

R. Takada, Y. Satomi, T. Kurata, N. Ueno, S. Norioka, H. Kondoh, T. Takao, S. Takada, Monounsaturated fatty acid modification of Wnt protein: Its role in Wnt secretion.
Dev. Cell
11
, 791-801 (2006).
[PubMed]

G. Hausmann, K. Basler, Wnt lipid modifications: Not as saturated as we thought.
Dev. Cell
11
, 751-752 (2006).
[PubMed]
Klotho Gets Around
Authors: Ray L. Bryan
Published: December 2006

A remarkable range of biological functions and mechanisms of action have been proposed for the protein Klotho. Klotho is a membrane protein with a large extracellular domain. However, its cytoplasmic domain includes just 11 amino acids, which are thought to be unlikely to mediate its regulatory functions. Lack of Klotho causes shortened life span in mice (but so might lots of deleterious mutations), and overexpression of the protein has been reported to extend life span. This was thought to reflect effects on signaling by insulin and insulin-like growth factor 1. Others have proposed that beta-glucuronidase activity of Klotho modifies the transient receptor potential ion channel 5 (TRPV5), supporting localization and activity of the latter at the plasma membrane. Klotho-deficient mice also share characteristics with mice lacking the fibroblast growth factor 23 (FGF23), such as infertility and decreased bone density; indeed, another proposed action of Klotho is the control of mineral metabolism. FGF23 also regulates mineral metabolism, and the latest action proposed for Klotho is to coordinate with the FGF receptor subtype IIIc [FGFR1 (IIIc)] to create a form of the FGFR that mediates binding of FGF23. Klotho turned up in a screen for molecules that bound in vitro to FGF23. FGF23 is itself unusual in that it functions like a hormone; that is, it is produced in bone but is carried in the circulation to regulate kidney function. Transfection of cells that also expressed FGFR1 (IIIc) with Klotho conferred specific high-affinity binding of FGF23 and allowed activation of associated signaling events. In vitro binding assays showed interaction of the extracellular domains of Klotho with a fusion protein of FGF23 with immunoglobulin Fc region. Injection of mice with a monoclonal antibody to Klotho caused changes reminiscent of those in Klotho knockout animals. Circulating FGF23 appears to act selectively at certain tissues, particularly the kidney, even though most tissues have FGFRs. The authors propose that the presence of Klotho may be the key factor that converts FGFR1(IIIc) to a receptor for FGF23, but they don’t propose a grander scheme to account for the range of reported actions of this intriguing protein.

I. Urakawa, Y. Yamazaki, T. Shimada, K. Iijima, H. Hasegawa, K. Okawa, T. Fujita, S. Fukumoto, T. Yamashita, Klotho converts canonical FGF receptor into a specific receptor for FGF23.
Nature
444
, 770-774 (2006).
[PubMed]
Regulatory RNAs
Authors: Riddihough Guy
Published: December 2006

Many small regulatory noncoding RNAs act by base-pairing with their target RNAs and interfering with their translation and/or affecting their stability, but not all such RNAs act in this way. The highly conserved prokaryotic 6
S
RNA interacts with and inhibits
Escherichia coli
σ70-containing RNA polymerase (RNAP). 6
S
RNA contains a single-stranded RNA bulge structure that mimics a gene promoter "open complex" and suggests that it might compete with promoters for RNAP. Wassarman and Saecker show that the bulge does indeed bind in the active site of RNAP in a manner analogous to the open complex, which prevents RNAP binding to legitimate targets. In the presence of nucleotides, the RNAP can synthesize short product RNAs from the 6
S
RNA template, which causes the RNAP to dissociate, freeing it to restart transcription during outgrowth from stationary phase and in response to nutrient availability.

K. M. Wassarman, R. M. Saecker, Synthesis-mediated release of a small RNA inhibitor of RNA polymerase.
Science
314
, 1601-1603 (2006).
[Abstract]
[Full Text]
Switching Neurotransmitter Effects in Development
Authors: Hines Pamela J.
Published: December 2006

The neurotransmitter GABA (γ-aminobutyric acid) generally exerts inhibitory effects on neuronal activity during adulthood, but during early development when circuits are being built, GABA has excitatory effects. Studying chick neurons, Liu
et al
. show that the change involves a switch in the direction of the chloride gradient across the cell membrane, which is in turn triggered by changes in nicotinic signaling activity. The change in signaling modality may reflect how neuronal activity and cellular development interact to fine-tune the structure of the brain.

Z. Liu, R. A. Neff, D. K. Berg, Sequential interplay of nicotinic and GABAergic signaling guides neuronal development.
Science
314
, 1610-1613 (2006).
[Abstract]
[Full Text]
From Oscillations to Patterning
Authors: Purnell Beverly
Published: December 2006

During early vertebrate development, blocks of mesodermal tissue, somites, are laid down on either side of the notochord in a periodic fashion following rhythmic waves of gene expression in the presomitic mesoderm. The somites subsequently give rise to skeletal muscle, axial skeleton, and part of the dermis. Dequéant
et al
. use a systematic analysis of genes expressed in the presomitic mesoderm over time. Oscillations of the fibroblast growth factor and Notch pathways alternated with components of the Wnt pathway, which suggests that an antagonism between these signaling pathways leads to the generation of phased somites.

M.-L. Dequéant, E. Glynn, K. Gaudenz, M. Wahl, J. Chen, A. Mushegian, O. Pourquié, A complex oscillating network of signaling genes underlies the mouse segmentation clock.
Science
314
, 1595-1598 (2006).
[Abstract]
[Full Text]
Understanding the Master Regulator
Authors: Ash Caroline
Published: December 2006

Bacterial pathogens need to be able to endure rapid and extreme environmental shifts and meanwhile ensure appropriate switches in gene expression. The two-component "master regulator" signaling system constituted by the PhoP regulator protein and the PhoQ Mg
2+
sensor is an important virulence regulator for pathogenic bacteria. Shin
et al
. found that immediately after activation of PhoP and PhoQ, an initial surge in transcription is followed by lower steady-state levels of transcription of target genes. The transcriptional surge was also observed in other two-component systems and may represent a general response that allows organisms to establish a new phenotype immediately in novel conditions.

D. Shin, E.-J. Lee, H. Huang, E. A. Groisman, A positive feedback loop promotes transcription surge that jump-starts
Salmonella
virulence circuit.
Science
314
, 1607-1609 (2006).
[Abstract]
[Full Text]
Intermediate Filaments as Signaling Platforms
Authors: Pallari Hanna-Mari , Eriksson John E.
Published: December 2006

Intermediate filaments (IFs) are cytoskeletal structures that are crucial for maintaining the structural and mechanical integrity of cells and tissues. Intriguingly, a wide range of previously unknown nonmechanical roles for the IF cytoskeleton are emerging: Recent studies have linked IFs to the integration of signals related to the determination of cell size, the regulation of cell migration and cell survival, and the buffering of the effects of stress-activated kinases. The characteristic structural features and expression patterns of the different members of this diverse family of highly abundant proteins make them well suited to act as cell- and tissue-specific modifiers and organizers of signaling.
Cytohesins Crucial for Hepatic Response to Insulin
Authors: Gough Nancy R.
Published: December 2006

Hafner
et al
. used an RNA aptamer displacement assay to identify new small molecule inhibitors of the guanine nucleotide exchange factor (GEF) family called cytohesins, which stimulate the activity of the ADP ribosylation factors (ARFs). The most potent and selective of these molecules was named SecinH3. Fuss
et al
. found that mutations in the
Drosophila
homolog of cytohesin, a protein called Steppke, caused a phenotype very similar to that seen with insulin signaling defects--small overall fly size at each stage of development but no impairment in eating. Feeding of SecinH3 to flies caused the same phenotype. Both the steppke-mutant flies and the SecinH3-treated flies exhibited biochemical and transcriptional evidence of loss of insulin signaling. Cytohesin appeared to act upstream of activation of phosphoinositide 3-kinase (PI3K), because the steppke-mutant phenotype was alleviated by expression of a constitutively active form of PI3K. Hafner
et al
. showed that SecinH3 inhibited insulin-dependent signaling in cultured HepG2 cells, a human liver cell line. Insulin also stimulated GTP loading of ARF6, which was blocked by SecinH3. Insulin signaling was blocked at the step of phosphorylation of insulin receptor substrate 1 (IRS1), but insulin receptor phosphorylation was not affected. Coimmunoprecipitation experiments showed that SecinH3 decreased the formation of an insulin-induced complex of cytohesin 2, cytohesin 3, ARF6, the insulin receptor, and IRS1. Mice fed SecinH3 exhibited signs of hepatic insulin resistance and increased circulating insulin, phenomena that are thought to cause type 2 diabetes in humans. Thus, alterations in cytohesin function may contribute to metabolic disorders (see Jackson for discussion).

M. Hafner, A. Schmitz, I. Grüne, S. G. Srivatsan, B. Paul, W. Kolanus, T. Quast, E. Kremmer, I. Bauer, M. Famulok, Inhibition of cytohesins by SecinH3 leads to hepatic insulin resistance.
Nature
444
, 941-944 (2006).
[PubMed]

B. Fuss, T. Becker, I. Zinke, M. Hoch, The cytohesin Steppke is essential for insulin signalling in
Drosophila
.
Nature
444
, 945-948 (2006).
[PubMed]

C. Jackson, Kicking off the insulin cascade.
Nature
444
, 833-834 (2006).
[PubMed]
The "Other" G Protein Corrals PKA
Authors: Adler Elizabeth M.
Published: December 2006

Whereas G
s
- and G
i
-type heterotrimeric GTP-binding proteins (G proteins) are well known for the stimulatory and inhibitory effects of their α-subunits on adenylyl cyclase, the known effects of G
o
--the most abundant brain G protein--are mostly mediated by the Gβγ dimer, with specific functions of Gα
o
remaining unclear. Noting that evidence exists both for direct effects of Gα
o
and for crosstalk between G
o
and cAMP-dependent protein kinase (PKA) signaling, Ghil
et al
. looked for possible interactions between G
o
and PKA. Both in vitro analysis and immunoprecipitation of 293T cells overexpressing Gα
o
and PKA regulatory and catalytic subunits (RIIβ and Cα) indicated that Gα
o
bound PKA catalytic subunits. Gα
i
did not bind Cα, and analysis of Gα
i
-Gα
o
chimeras indicated that Gα
o
bound PKA through its GTPase domain. Gα
o
did not inhibit the catalytic activity of purified Cα; however, immunocytochemical analysis of COS7 cells transfected with FLAG-tagged Gα constructs and stimulated with forskolin indicated that the Gα
o
-Cα interaction inhibited translocation of Cα to the nucleus. Moreover, pharmacological analysis of GH4C1 rat pituitary tumor cells (containing endogenous G
o
and PKA) indicated that G
o
inhibited not only Cα translocation to the nucleus but also PKA-dependent cAMP response element-binding protein (CREB) phosphorylation. In contrast, Gα
ο
spared--or even facilitated--the cytosolic effects of PKA. Thus, the authors propose that, by binding Cα, Gα
o
directs its subcellular localization and thereby regulates its downstream effects.

S. Ghil, J.-M. Choi, S.-S. Kim, Y.-D. Lee, Y. Liao, L. Birnbaumer, H. Suh-Kim, Compartmentalization of protein kinase A signaling by the heterotrimeric G protein G
o
.
Proc. Natl. Acad. Sci. U.S.A.
103
, 19158-19163 (2006).
[Abstract]
[Full Text]
Will Heteromeric G Protein-Coupled Receptors Confound Drug Development?
Authors: Ray L. Bryan
Published: December 2006

Although the jury is still out, evidence is accumulating that heterotrimeric guanine nucleotide-binding protein-coupled receptors (GPCRs) may function as dimers or oligomers. Even though much of the evidence is from experiments in which the receptors are heterologously expressed in transfected cells, the findings merit attention because of their implications for drug discovery (GPCRs being a major focus of the efforts of the pharmaceutical industry). Ellis
et al
. provide evidence for interaction of two GPCRs, the human cannabinoid CB1 receptor and the receptor for orexins (peptides that stimulate eating). The orexin-1 receptor was localized primarily at the cell surface in transfected human embryonic kidney (HEK) 293 cells, but when cells were also transfected with the CB1 receptor, orexin-1 receptors localized intracellularly, as did CB1 receptors when transfected alone. Single-cell FRET (fluorescence resonance energy transfer) imaging in cells expressing appropriately tagged receptors showed that the receptors indicated the close apposition of the expressed receptors at intracellular sites. The key finding, though, was that specific antagonists of either the CB1 receptor or orexin A receptors could alter the response of the other receptor to its normal ligand. In cells expressing both receptors, treatment with the CB1 receptor agonist caused both receptors to relocalize to the cell surface but reduced the potency of orexin A to produce changes in activity of mitogen-activated protein (MAP) kinases. Similarly, treatment of cells expressing both receptors with an orexin receptor antagonist reduced the potency of a CB1 receptor agonist to regulate MAP kinase activity. The authors point out that if such receptor interactions exist in vivo, their properties may substantially influence the specificity of agents designed for therapeutic purposes. The effects described in the current paper provide a means for ligands that have no affinity for a particular receptor to still alter the signaling properties of that receptor by influencing the localization and signaling of heterodimeric receptors.

J. Ellis, J. D. Pediani, M. Canals, S. Milasta, G. Milligan, Orexin-1 receptor-cannabinoid CB1 receptor heterodimerization results in both ligand-dependent and -independent coordinated alterations of receptor localization and function.
J. Biol. Chem.
281
, 38812-38824 (2006).
[Abstract]
[Full Text]
Climbing the Gradient
Authors: Ray L. Bryan
Published: December 2006

During chemotaxis, cells respond to tiny changes in the concentration of chemoattractant molecules and move toward their source. Chen
et al
. (see the Perspective by Linden) show that in addition to receptors for the chemoattractant peptide
N
-formyl-Met-Leu-Phe (fMLP), human neutrophils use two other receptor systems to promote appropriate cell migration. Neutrophils exposed to a concentration gradient of fMLP released adenosine triphosphate (ATP) at the leading edge of the cell. The released ATP appeared to act in an autocrine manner and stimulated purinergic receptors to provide a signal required for proper orientation of the cell.

Y. Chen, R. Corriden, Y. Inoue, L. Yip, N. Hashiguchi, A. Zinkernagel, V. Nizet, P. A. Insel, W. G. Junger, ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors.
Science
314
, 1792-1795 (2006).
[Abstract]
[Full Text]

J. Linden, Purinergic chemotaxis.
Science
314
, 1689-1690 (2006).
[Abstract]
[Full Text]
Injectable Virulence
Authors: Ash Caroline
Published: December 2006

Little is known about the molecular determinants of virulence in eukaryotic pathogens like
Toxoplasma gondii
and malaria. Progress has been hampered by inefficient genetic tools, large genomes, and complex life cycles. Using forward genetic analysis, Taylor
et al
. and Saeij
et al
. show that a few clustered genes on a single chromosome control the dramatic difference seen in the virulence of natural lineages of the parasite
T. gondii
. The most important of these genes encodes a conserved serine/threonine kinase that is injected into the host cell. Although this process is reminiscent of type III secretion in bacteria, it is mechanistically and evolutionarily distinct.

S. Taylor, A. Barragan, C. Su, B. Fux, S. J. Fentress, K. Tang, W. L. Beatty, H. El Hajj, M. Jerome, M. S. Behnke, M. White, J. C. Wootton, L. D. Sibley, A secreted serine-threonine kinase determines virulence in the eukaryotic pathogen
Toxoplasma gondii
.
Science
314
, 1776-1780 (2006).
[Abstract]
[Full Text]

J. P. J. Saeij, J. P. Boyle, S. Coller, S. Taylor, L. D. Sibley, E. T. Brooke-Powell, J. W. Ajioka, J. C. Boothroyd, Polymorphic secreted kinases are key virulence factors in toxoplasmosis.
Science
314
, 1780-1783 (2006).
[Abstract]
[Full Text]
α-Proteobacterial Symbionts of Marine Bryozoans in the GenusWatersipora
Authors: Anderson Christine M. , Haygood Margo G.
Published: December 2006

Bacterial symbionts that resembled mollicutes were discovered in the marine bryozoanWatersipora arcuatain the 1980s. In this study, we used PCR and sequencing of 16S rRNA genes, specific fluorescence in situ hybridization, and phylogenetic analysis to determine that the bacterial symbionts of “W. subtorquata” and “W. arcuata” from several locations along the California coast are actually closely related α-Proteobacteria, not mollicutes. We propose the names“CandidatusEndowatersipora palomitas” and “CandidatusEndowatersipora rubus” for the symbionts of “W. subtorquata” and “W. arcuata,” respectively.
Mutation of
Candida tropicalis
by Irradiation with a He-Ne Laser To Increase Its Ability To Degrade Phenol
Authors: Jiang Yan , Wen Jianping , Jia Xiaoqiang , Caiyin Qinggele , Hu Zongding
Published: December 2006

Candida tropicalis
isolated from acclimated activated sludge was used in this study. Cell suspensions with 5 × 10
7
cells ml
−1
were irradiated by using a He-Ne laser. After mutagenesis, the irradiated cell suspension was diluted and plated on yeast extract-peptone-dextrose (YEPD) medium. Plates with approximately 20 individual colonies were selected, and all individual colonies were harvested for phenol biodegradation. The phenol biodegradation stabilities for 70 phenol biodegradation-positive mutants, mutant strains CTM 1 to 70, ranked according to their original phenol biodegradation potentials, were tested continuously during transfers. Finally, mutant strain CTM 2, which degraded 2,600 mg liter
−1
phenol within 70.5 h, was obtained on the basis of its capacity and hereditary stability for phenol biodegradation. The phenol hydroxylase gene sequences were cloned in wild and mutant strains. The results showed that four amino acids were mutated by irradiation with a laser. In order to compare the activity of phenol hydroxylase in wild and mutant strains, their genes were expressed in
Escherichia coli
BL21(DE3) and enzyme activities were spectrophotometrically determined. It was clear that the activity of phenol hydroxylase was promoted after irradiation with a He-Ne laser. In addition, the cell growth and intrinsic phenol biodegradation kinetics of mutant strain CTM 2 in batch cultures were also described by Haldane's kinetic equation with a wide range of initial phenol concentrations from 0 to 2,600 mg liter
−1
. The specific growth and degradation rates further demonstrated that the CTM 2 mutant strain possessed a higher capacity to resist phenol toxicity than wild
C. tropicalis
did.
Identification of
Escherichia coli
O157:H7 Genomic Regions Conserved in Strains with a Genotype Associated with Human Infection
Authors: Steele Marina , Ziebell Kim , Zhang Yongxiang , Benson Andrew , Konczy Paulina , Johnson Roger , Gannon Victor
Published: December 2006

Beta-glucuronidase-negative, sorbitol-nonfermenting isolates of Shiga toxin-producing
Escherichia coli
O157 comprise part of a clone complex of related enterohemorrhagic
E. coli
isolates. High-resolution genotyping shows that the O157 populations have diverged into two different lineages that appear to have different ecologies. To identify genomic regions unique to the most common human-associated genotype, suppression subtractive hybridization was used to identify DNA sequences present in two clinical strains representing the human lineage I O157:H7 strains but absent from two bovine-derived lineage II strains. PCR assays were then used to test for the presence of these regions in 10 lineage I strains and 20 lineage II strains. Twelve conserved regions of genomic difference for lineage I (CRD
I
) were identified that were each present in at least seven of the lineage I strains but absent in most of the lineage II strains tested. The boundaries of the lineage I conserved regions were further delimited by PCR. Eleven of these CRD
I
were associated with
E. coli
Sakai S-loops 14, 16, 69, 72, 78, 82, 83, 91 to 93, 153, and 286, and the final CRD
I
was located on the pO157 virulence plasmid. Several potential virulence factors were identified within these regions, including a putative hemolysin-activating protein, an iron transport system, and several possible regulatory genes. Cluster analysis based on lineage I conserved regions showed that the presence/absence of these regions was congruent with the inferred phylogeny of the strains.
Comparative Analysis of Virulence Genes, Genetic Diversity, and Phylogeny of Commensal and Enterotoxigenic
Escherichia coli
Isolates from Weaned Pigs
Authors: Wu Xi-Yang , Chapman Toni , Trott Darren J. , Bettelheim Karl , Do Thuy N. , Driesen Steve , Walker Mark J. , Chin James
Published: December 2006

If the acquisition of virulence genes (VGs) for pathogenicity were not solely acquired through horizontal gene transfers of pathogenicity islands, transposons, and phages, then clonal clusters of enterotoxigenic
Escherichia coli
(ETEC) would contain few or even none of the VGs found in strains responsible for extraintestinal infections. To evaluate this possibility, 47 postweaning diarrhea (PWD) ETEC strains from different geographical origins and 158 commensal
E. coli
isolates from the gastrointestinal tracts of eight group-housed healthy pigs were screened for 36 extraintestinal and 18 enteric VGs using multiplex PCR assays. Of 36 extraintestinal VGs, only 8 were detected (
fimH
,
traT
,
fyuA
,
hlyA
,
kpsMtII
,
k5
,
iha
, and
ompT
) in the ETEC collection. Among these,
hlyA
(α-hemolysin) and
iha
(nonhemagglutinating adhesin) occurred significantly more frequently among the ETEC isolates than in the commensal isolates. Clustering analysis based on the VG profiles separated commensal and ETEC isolates and even differentiated serogroup O141 from O149. On the other hand, pulsed-field gel electrophoresis (PFGE) successfully clustered ETEC isolates according to both serotype and geographical origin. In contrast, the commensal isolates were heterogeneous with respect to both serotype and DNA fingerprint. This study has validated the use of VG profiling to examine pathogenic relationships between porcine ETEC isolates. The clonal relationships of these isolates can be further clarified by PFGE fingerprinting. The presence of extraintestinal VGs in porcine ETEC confirmed the hypothesis that individual virulence gene acquisitions can occur concurrently against a background of horizontal gene transfers of pathogenicity islands. Over time, this could enable specific clonotypes to respond to host selection pressure and to evolve into new strains with increased virulence.
Use of 16S rRNA and
rpoB
Genes as Molecular Markers for Microbial Ecology Studies
Authors: Case Rebecca J. , Boucher Yan , Dahllöf Ingela , Holmström Carola , Doolittle W. Ford , Kjelleberg Staffan
Published: December 2006

Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene
rpoB
. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such “heterogeneity hot spots” occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below.
rpoB
provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as
rpoB
could complement the information provided by the 16S rRNA gene.
Sensitive Quantitative Detection of Commensal Bacteria by rRNA-Targeted Reverse Transcription-PCR
Authors: Matsuda Kazunori , Tsuji Hirokazu , Asahara Takashi , Kado Yukiko , Nomoto Koji
Published: December 2006

A sensitive rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) method was developed for exact and sensitive enumeration of subdominant bacterial populations. Using group- or species-specific primers for 16S or 23S rRNA, analytical curves were constructed for
Escherichia coli
,
Enterococcus faecalis
,
Staphylococcus aureus
,
Clostridium perfringens
, and
Pseudomonas aeruginosa
, and the threshold cycle value was found to be linear up to an RNA amount of 10
−3
cell per RT-PCR. The number of bacteria in culture was determined by RT-qPCR, and the results correlated well with the CFU count over the range from 10
0
to 10
5
CFU. The bacterial counts obtained by RT-qPCR were the same as the CFU counts irrespective of the growth phase in vitro, except for
C. perfringens
during starvation periods; the viable cell counts obtained by using a combination of 4′,6-diamidino-2-phenylindole (DAPI) staining and SYTO9-propidium iodide double staining were in good agreement with the RT-qPCR counts rather than with the CFU counts. The RT-qPCR method could detect endogenous
Enterobacteriaceae
and
P. aeruginosa
in feces of hospitalized patients (
n
= 38) at a level of 10
3
cells per g of feces, and for enumeration of
S. aureus
or
P. aeruginosa
spiked into human peripheral blood, the lower detection limit for RT-qPCR quantification of the bacteria was 2 cells per ml of blood, suggesting that this method was equivalent to the conventional culture method. As only 5 h was needed for RT-qPCR quantification, we suggest that rRNA-targeted RT-qPCR assays provide a sensitive and convenient system for quantification of commensal bacteria and for examining their possible invasion of a host.
Procedure for Rapid Concentration and Detection of Enteric Viruses from Berries and Vegetables
Authors: Butot S. , Putallaz T. , Sánchez G.
Published: December 2006

Several hepatitis A virus (HAV) and norovirus (NV) outbreaks due to consumption of berries and vegetables have been reported during recent years. To facilitate the detection of enteric viruses that may be present on different fresh and frozen products, we developed a rapid and sensitive detection method for HAV, NV, and rotavirus (RV). Initial experiments focused on optimizing the composition of the elution buffer, improving the viral concentration method, and evaluating the performance of various extraction kits. Viruses were extracted from the food surface by a direct elution method in a glycine-Tris (pH 9.5) buffer containing 1% beef extract and concentrated by ultrafiltration. Occasionally, PCR inhibitors were present in the processed berry samples, which gave relatively poor detection limits. However, this problem was overcome by adding a pectinase treatment in the protocol, which markedly improved the sensitivity of the method. After optimization, this concentration method was applied in combination with real-time reverse transcription-PCR (RT-PCR) using specific primers in various types of berries and vegetables. The average detection limits were 1 50% tissue culture infective dose (TCID
50
), 54 RT-PCR units, and 0.02 TCID
50
per 15 g of food for HAV, NV, and RV, respectively. Based on our results, it is concluded that this procedure is suitable to detect and quantify enteric viruses within 6 h and can be applied for surveillance of enteric viruses in fresh and frozen products.
Efficient Production of
l
-Lactic Acid from Xylose by
Pichia stipitis
Authors: Ilmén Marja , Koivuranta Kari , Ruohonen Laura , Suominen Pirkko , Penttilä Merja
Published: December 2006

Microbial conversion of renewable raw materials to useful products is an important objective in industrial biotechnology.
Pichia stipitis
, a yeast that naturally ferments xylose, was genetically engineered for
l
-(+)-lactate production. We constructed a
P. stipitis
strain that expressed the
l
-lactate dehydrogenase (LDH) from
Lactobacillus helveticus
under the control of the
P. stipitis
fermentative
ADH1
promoter. Xylose, glucose, or a mixture of the two sugars was used as the carbon source for lactate production. The constructed
P. stipitis
strain produced a higher level of lactate and a higher yield on xylose than on glucose. Lactate accumulated as the main product in xylose-containing medium, with 58 g/liter lactate produced from 100 g/liter xylose. Relatively efficient lactate production also occurred on glucose medium, with 41 g/liter lactate produced from 94 g/liter glucose. In the presence of both sugars, xylose and glucose were consumed simultaneously and converted predominantly to lactate. Lactate was produced at the expense of ethanol, whose production decreased to ∼15 to 30% of the wild-type level on xylose-containing medium and to 70 to 80% of the wild-type level on glucose-containing medium. Thus, LDH competed efficiently with the ethanol pathway for pyruvate, even though the pathway from pyruvate to ethanol was intact. Our results show, for the first time, that lactate production from xylose by a yeast species is feasible and efficient. This is encouraging for further development of yeast-based bioprocesses to produce lactate from lignocellulosic raw material.
Shiga Toxin and Shiga Toxin-Encoding Phage Do Not Facilitate
Escherichia coli
O157:H7 Colonization in Sheep
Authors: Cornick Nancy A. , Helgerson Amy F. , Sharma Vijay
Published: December 2006

Isogenic strains of
Escherichia coli
O157:H7, missing either
stx
2
or the entire Stx2-encoding phage, were compared with the parent strain for their abilities to colonize sheep. The absence of the phage or of the Shiga toxin did not significantly impact the magnitude or duration of shedding of
E. coli
O157:H7.