bluuebunny/crossref_metadata_2025 huggingface dataset:
Contributions of ATP, GTP, and Redox State to Nutritional Stress Activation of theBacillus subtilisσBTranscription Factor
Authors: Zhang Shuyu , Haldenwang W. G.
Published: November 2005

The general stress regulon ofBacillus subtilisis induced by activation of the σBtranscription factor. σBactivation occurs when one of two phosphatases responds to physical or nutritional stress to activate a positive σBregulator by dephosphorylation. The signal that triggers the nutritional stress phosphatase (RsbP) is unknown; however, RsbP activation occurs under culture conditions (glucose/phosphate starvation, azide or decoyinine treatment) that reduce the cell's levels of ATP and/or GTP. Variances in nucleotide levels in these instances may be coincidental rather than causal. RsbP carries a domain (PAS) that in some regulatory systems can respond directly to changes in electron transport, proton motive force, or redox potential, changes that typically precede shifts in high-energy nucleotide levels. The current work usesBacillus subtiliswith mutations in the oxidative phosphorylation and purine nucleotide biosynthetic pathways in conjunction with metabolic inhibitors to better define the inducing signal for RsbP activation. The data argue that a drop in ATP, rather than changes in GTP, proton motive force, or redox state, is the key to triggering σBactivation.
Adaptations of
Pseudomonas aeruginosa
to the Cystic Fibrosis Lung Environment Can Include Deregulation of
zwf
, Encoding Glucose-6-Phosphate Dehydrogenase
Authors: Silo-Suh Laura , Suh Sang-Jin , Phibbs Paul V. , Ohman Dennis E.
Published: November 2005

Cystic fibrosis (CF) patients are highly susceptible to chronic pulmonary disease caused by mucoid
Pseudomonas aeruginosa
strains that overproduce the exopolysaccharide alginate. We showed here that a mutation in
zwf
, encoding glucose-6-phosphate dehydrogenase (G6PDH), leads to a ∼90% reduction in alginate production in the mucoid, CF isolate,
P. aeruginosa
FRD1. The main regulator of alginate, sigma-22 encoded by
algT
(
algU
), plays a small but demonstrable role in the induction of
zwf
expression in
P. aeruginosa
. However, G6PDH activity and
zwf
expression were higher in FRD1 strains than in PAO1 strains. In PAO1,
zwf
expression and G6PDH activity are known to be subject to catabolite repression by succinate. In contrast, FRD1
zwf
expression and G6PDH activity were shown to be refractory to such catabolite repression. This was apparently not due to a defect in the catabolite repression control (Crc) protein. Such relaxed control of
zwf
was found to be common among several examined CF isolates but was not seen in other strains of clinical and environmental origin. Two sets of clonal isolates from individual CF patient indicated that the resident
P. aeruginosa
strain underwent an adaptive change that deregulated
zwf
expression. We hypothesized that high-level, unregulated G6PDH activity provided a survival advantage to
P. aeruginosa
within the lung environment. Interestingly,
zwf
expression in
P. aeruginosa
was shown to be required for its resistance to human sputum. This study illustrates that adaptation to the CF pulmonary environment by
P. aeruginosa
can include altered regulation of basic metabolic activities, including carbon catabolism.
Periplasmic Peptidyl Prolyl
cis-trans
Isomerases Are Not Essential for Viability, but SurA Is Required for Pilus Biogenesis in
Escherichia coli
Authors: Justice Sheryl S. , Hunstad David A. , Harper Jill Reiss , Duguay Amy R. , Pinkner Jerome S. , Bann James , Frieden Carl , Silhavy Thomas J. , Hultgren Scott J.
Published: November 2005

In
Escherichia coli
, FkpA, PpiA, PpiD, and SurA are the four known periplasmic
cis
-
trans
prolyl isomerases. These isomerases facilitate proper protein folding by increasing the rate of transition of proline residues between the
cis
and
trans
states. Genetic inactivation of all four periplasmic isomerases resulted in a viable strain that exhibited a decreased growth rate and increased susceptibility to certain antibiotics. Levels of the outer membrane proteins LamB and OmpA in the quadruple mutant were indistinguishable from those in the
surA
single mutant. In addition, expression of P and type 1 pili (adhesive organelles produced by uropathogenic strains of
E. coli
and assembled by the chaperone/usher pathway) were severely diminished in the absence of the four periplasmic isomerases. Maturation of the usher was significantly impaired in the outer membranes of strains devoid of all four periplasmic isomerases, resulting in a defect in pilus assembly. Moreover, this defect in pilus assembly and usher stability could be attributed to the absence of SurA. The data presented here suggest that the four periplasmic isomerases are not essential for growth under laboratory conditions but may have significant roles in survival in environmental and pathogenic niches, as indicated by the effect on pilus production.
Diverse Phenotypes Resulting from Polyphosphate Kinase Gene (
ppk1
) Inactivation in Different Strains of
Helicobacter pylori
Authors: Tan Shumin , Fraley Cresson D. , Zhang Maojun , Dailidiene Daiva , Kornberg Arthur , Berg Douglas E.
Published: November 2005

Connections among biochemical pathways should help buffer organisms against environmental stress and affect the pace and trajectory of genome evolution. To explore these ideas, we studied consequences of inactivating the gene for polyphosphate kinase 1 (
ppk1
) in strains of
Helicobacter pylori
, a genetically diverse gastric pathogen. The PPK1 enzyme catalyzes synthesis of inorganic polyphosphate (poly P), a reservoir of high-energy phosphate bonds with multiple roles. Prior analyses in less-fastidious microbes had implicated poly P in stress resistance, motility, and virulence. In our studies,
ppk1
inactivation caused the expected near-complete absence of poly P (>250-fold decrease) but had phenotypic effects that differed markedly among unrelated strains: (i) poor initial growth on standard brain heart infusion agar (five of six strains tested); (ii) weakened colonization of mice (4 of 5 strains); (iii) reduced growth on Ham's F-12 agar, a nutritionally limiting medium (8 of 11 strains); (iv) heightened susceptibility to metronidazole (6 of 17 strains); and (v) decreased motility in soft agar (1 of 13 strains). Complementation tests confirmed that the lack of growth of one Δ
ppk1
strain on F-12 agar and the inability to colonize mice of another were each due to
ppk1
inactivation. Thus, the importance of
ppk1
to
H. pylori
differed among strains and the phenotypes monitored. We suggest that quantitative interactions, as seen here, are common among genes that affect metabolic pathways and that
H. pylori
's high genetic diversity makes it well suited for studies of such interactions, their underlying mechanisms, and their evolutionary consequences.
A Gyrase Mutant with Low Activity Disrupts Supercoiling at the Replication Terminus
Authors: Pang Zhenhua , Chen Ray , Manna Dipankar , Higgins N. Patrick
Published: November 2005

When a mutation in an essential gene shows a temperature-sensitive phenotype, one usually assumes that the protein is inactive at nonpermissive temperature. DNA gyrase is an essential bacterial enzyme composed of two subunits, GyrA and GyrB. ThegyrB652mutation results from a single base change that substitutes a serine residue for arginine 436 (R436-S) in the GyrB protein. At 42°C, strains with thegyrB652allele stop DNA replication, and at 37°C, such strains grow but have RecA-dependent SOS induction and show constitutive RecBCD-dependent DNA degradation. Surprisingly, the GyrB652 protein is not inactive at 42°C in vivo or in vitro and it doesn't directly produce breaks in chromosomal DNA. Rather, this mutant has a lowkcatcompared to wild-type GyrB subunit. With more than twice the normal mean number of supercoil domains, this gyrase hypomorph is prone to fork collapse and topological chaos near the terminus of DNA replication.
Novel Virulence Gene of
Pseudomonas syringae
pv. tomato Strain DC3000
Authors: Preiter Karen , Brooks David M. , Penaloza-Vazquez Alejandro , Sreedharan Aswathy , Bender Carol L. , Kunkel Barbara N.
Published: November 2005

Previously, we conducted a mutant screen of
Pseudomonas syringae
pv. tomato strain DC3000 to identify genes that contribute to virulence on
Arabidopsis thaliana
plants. Here we describe the characterization of one mutant strain, DB4H2, which contains a single Tn
5
insertion in PSPTO3576, an open reading frame that is predicted to encode a protein belonging to the TetR family of transcriptional regulators. We demonstrate that PSPTO3576 is necessary for virulence in DC3000 and designate the encoded protein TvrR (
T
etR-like
v
i
r
ulence
r
egulator). TvrR, like many other TetR-like transcriptional regulators, negatively regulates its own expression. Despite the presence of a putative HrpL binding site in the
tvrR
promoter region,
tvrR
is not regulated by HrpL, an alternative sigma factor that regulates the expression of many known DC3000 virulence genes.
tvrR
mutant strains grow comparably to wild-type DC3000 in culture and possess an intact type III secretion system. However,
tvrR
mutants do not cause disease symptoms on inoculated
A. thaliana
and tomato plants, and their growth within plant tissue is significantly impaired. We demonstrate that
tvrR
mutant strains are able to synthesize coronatine (COR), a phytotoxin required for virulence of DC3000 on
A. thaliana
. Given that
tvrR
mutant strains are not defective for type III secretion or COR production,
tvrR
appears to be a novel virulence factor required for a previously unexplored process that is necessary for pathogenesis.
Polar Localization of a Soluble Methyl-Accepting Protein ofPseudomonas aeruginosa
Authors: Bardy Sonia L. , Maddock Janine R.
Published: November 2005

A soluble methyl-accepting chemotaxis protein (MCP) ofPseudomonas aeruginosa, McpS, showed polar localization by immunofluorescence microscopy. Overexpression of McpS resulted in a dominant-negative effect on chemotaxis and caused a loss of polar clustering of the general MCP population. The polar localization of a soluble MCP defines a third, and unexpected, paradigm for cellular MCP localization.
Streptococcus mutans
Murein Hydrolase
Authors: Catt Diana M. , Gregory Richard L.
Published: November 2005

Allelic replacement of the C terminus of a
Streptococcus mutans
surface protein affects murein hydrolase activity. The targeted open reading frame encodes a 67-kDa protein (SmaA) with an N-terminal signal sequence and cleavage site, three 46-amino-acid (aa) direct repeats, and two 88-aa direct repeats. The identical autolytic profile was obtained using a sortase mutant (SrtA
−
).
TNF-α dilates cerebral arteries via NAD(P)H oxidase-dependent Ca2+ spark activation
Authors: Cheranov Sergey Y. , Jaggar Jonathan H.
Published: November 2005

Expression of TNF-α, a pleiotropic cytokine, is elevated during stroke and cerebral ischemia. TNF-α regulates arterial diameter, although mechanisms mediating this effect are unclear. In the present study, we tested the hypothesis that TNF-α regulates the diameter of resistance-sized (∼150-μm diameter) cerebral arteries by modulating local and global intracellular Ca2+ signals in smooth muscle cells. Laser-scanning confocal imaging revealed that TNF-α increased Ca2+ spark and Ca2+ wave frequency but reduced global intracellular Ca2+ concentration ([Ca2+]i) in smooth muscle cells of intact arteries. TNF-α elevated reactive oxygen species (ROS) in smooth muscle cells of intact arteries, and this increase was prevented by apocynin or diphenyleneiodonium (DPI), both of which are NAD(P)H oxidase blockers, but was unaffected by inhibitors of other ROS-generating enzymes. In voltage-clamped (−40 mV) cells, TNF-α increased the frequency and amplitude of Ca2+ spark-induced, large-conductance, Ca2+-activated K+ (KCa) channel transients ∼1.7- and ∼1.4-fold, respectively. TNF-α-induced transient KCa current activation was reversed by apocynin or by Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a membrane-permeant antioxidant, and was prevented by intracellular dialysis of catalase. TNF-α induced reversible and similar amplitude dilations in either endothelium-intact or endothelium-denuded pressurized (60 mmHg) cerebral arteries. MnTMPyP, thapsigargin, a sarcoplasmic reticulum Ca2+-ATPase blocker that inhibits Ca2+ sparks, and iberiotoxin, a KCa channel blocker, reduced TNF-α-induced vasodilations to between 15 and 33% of control. In summary, our data indicate that TNF-α activates NAD(P)H oxidase, resulting in an increase in intracellular H2O2 that stimulates Ca2+ sparks and transient KCa currents, leading to a reduction in global [Ca2+]i, and vasodilation.
Identification and characterization of novel IGFBP5 interacting protein: evidence IGFBP5-IP is a potential regulator of osteoblast cell proliferation
Authors: Amaar Yousef G. , Tapia Blanca , Chen Shin-Tai , Baylink David J. , Mohan Subburaman
Published: November 2005

Insulin-like growth factor binding protein-5 (IGFBP5) is a multifunctional protein, which acts not only as a traditional binding protein, but also functions as a growth factor independent of IGFs to stimulate bone formation. It has been predicted that the intrinsic growth factor action of IGFBP5 involves binding of IGFBP5 to a putative receptor to induce downstream signaling pathways and/or nuclear translocation of IGFBP5 to influence transcription of genes involved in osteoblast cell proliferation/differentiation. Our study indentified proteins that bound to IGFBP5 using IGFBP5 as bait in a yeast two-hybrid screen of the U2 human osteosarcoma cell cDNA library. One of the clones that interacted strongly with the bait under high-stringency conditions corresponded to a novel IGFBP5 interacting protein (IGFBP5-IP) encoded by a gene that resides in mouse chromosome 10. The interaction between IGFBP5-IP and IGFBP5 is confirmed by in vitro coimmunoprecipitation studies that used pFlag and IGFBP5 polyclonal antibody, and cell lysates overexpressing both IGFBP5-IP and IGFBP5. Northern blot and RT-PCR analysis showed that the IGFBP-IP is expressed in both untransformed normal human osteoblasts and in osteosarcoma cell lines, which are known to produce IGFBP5. To determine the roles of IGFBP5-IP, we evaluated the effect of blocking the expression of IGFBP5-IP on osteoblast proliferation. We found that using a IGFBP5-IP-specific small interfering-hairpin plasmid resulted in a decrease in both basal and IGFBP5-induced osteoblast cell proliferation. On the basis of these findings, we predict that IGFBP5-IP may act as intracellular mediator of growth promoting actions of IGFBP5 and perhaps other osteoregulatory agents in bone cells.
11β-Hydroxysteroid dehydrogenase type 2 and the regulation of surfactant protein A by dexamethasone metabolites
Authors: Garbrecht Mark R. , Schmidt Thomas J. , Krozowski Zygmunt S. , Snyder Jeanne M.
Published: November 2005

Glucocorticoid (GC) metabolism by the 11β-hydroxysteroid dehydrogenase (HSD) system is an important prereceptor regulator of GC action. The HSD enzymes catalyze the interconversion of the endogenous, biologically active GC cortisol and its inactive 11-dehydro metabolite cortisone. The role of the HSD enzymes in the metabolism of synthetic GCs, such as dexamethasone (Dex), is more complex. The human lung is a classic GC-sensitive organ; however, the roles of the HSD enzymes (HSD1 and HSD2) in the human lung are poorly understood. In the present study, we examined the expression of the HSD enzymes in human adult and fetal lung tissues and the human lung epithelial cell line NCI-H441. We observed that human adult and fetal lung tissues, as well as H441 cells, express HSD2 protein and that it is upregulated by Dex (10−7M). By contrast, HSD1 protein was undetectable. We also show that the Dex-mediated regulation of surfactant protein A is attenuated by inhibition of HSD2 activity. Furthermore, we demonstrate that unlike the inactive, 11-dehydro metabolite of cortisol (i.e., cortisone), the 11-dehydro metabolite of Dex, 11-dehydro-Dex, competes for binding to the GC receptor (GR) in human lung epithelial cells and retains GR agonist activity. Together, these data suggest that differences exist in the biological activities of the metabolites of cortisol and Dex.
Nitric oxide synthase inhibition activates L- and T-type Ca2+channels in afferent and efferent arterioles
Authors: Feng Ming-Guo , Navar L. Gabriel
Published: November 2005

Previous studies have shown that L-type Ca2+channel (LCC) blockers primarily dilate resting and ANG II-constricted afferent arterioles (AA), but do not influence either resting or ANG II-constricted efferent arterioles (EA). In contrast, blockade of T-type Ca2+channels (TCC) dilate EA and prevent ANG II-mediated efferent constriction. The present study determined the role of LCC and TCC in mediating the AA and EA constriction following inhibition of nitric oxide synthase (NOS) and tested the hypothesis that inhibition of NOS increases the influence of LCC on EA. With the use of an isolated blood-perfused rat juxtamedullary nephron preparation, single AA or EA were visualized and superfused with a NOS inhibitor, N-nitro-l-arginine (l-NNA), with or without concomitant treatment with an LCC blocker, diltiazem, or a TCC blocker, pimozide. In response to l-NNA (1, 10, and 100 μmol/l), AA and EA diameters decreased significantly by 6.0 ± 0.3, 13.7 ± 1.7, and 19.9 ± 1.4%, and by 6.2 ± 0.5, 13.3 ± 1.1, and 19.0 ± 1.9%, respectively. During TCC blockade with pimozide (10 μmol/l), l-NNA did not significantly constrict afferent (0.9 ± 0.6, 1.5 ± 0.5, and 1.7 ± 0.5%) or efferent (0.4 ± 0.1, 2.1 ± 0.7, and 2.5 ± 1.0%) arterioles. In contrast to the responses with other vasoconstictors, the l-NNA-induced constriction of EA, as well as AA, was reversed by diltiazem (10 μmol/l). The effects were overlapping as pimozide superimposed on diltiazem did not elicit further dilation. When the effects of l-NNA were reversed by superfusion with an NO donor, SNAP (10 μmol/l), diltiazem did not cause significant efferent dilation. As a further test of LCC activity, 55 mmol/l KCl, which depolarizes and constricts AA, caused only a modest constriction in resting EA (8.7 ± 1.3%), but a stronger EA constriction during concurrent treatment with l-NNA (23.8 ± 4.8%). In contrast, norepinephrine caused similar constrictions in both l-NNA-treated and nontreated arterioles. These results provide evidence that NO inhibits LCC and TCC activity and that NOS inhibition-mediated arteriolar constriction involves activation of LCC and TCC in both AA and EA. The difference in responses to high KCl between resting and l-NNA-constricted EA and the ability of diltiazem to block EA constriction caused by l-NNA contrasts with the lack of efferent effects in resting and SNAP-treated l-NNA-preconstricted arterioles and during ANG II-mediated vasoconstriction, suggesting a recruitment of LCC in EA when NOS is inhibited. These data help explain how endothelial dysfunction associated with hypertension may lead to enhanced activity of LCC in postglomerular arterioles and increased postglomerular resistance.
Synapse Elimination in the Corticospinal Projection During the Early Postnatal Period
Authors: Kamiyama Tsutomu , Yoshioka Noboru , Sakurai Masaki
Published: November 2005

In corticospinal synapses reconstructed in vitro by slice co-culture, we previously showed that the synapses were distributed across the gray matter at 6–7 days in vitro (DIV). Thereafter, they began to be eliminated from the ventral side, and dorsal-dominant distribution was nearly complete at 11–12 DIV. The synapse elimination is associated with retraction of the corticospinal (CS) terminals. We studied whether this specific type of synapse elimination is a physiological phenomenon rather than in vitro artifact. The rat corticospinal tract was stimulated at the medullary pyramid, and field potentials were recorded at the cervical cord along an 200-μm interval lattice on the axial plane. Clearly defined negative field potential were identified as field excitatory postsynaptic potentials (fEPSPs) generated by corticospinal synapses. They were recorded from the entire spinal gray matter at postnatal day 7 (P7). These negative fEPSPs reversed to positive in the most ventrolateral part at P8. Reversal extended to the more mediodorsal area at P10, indicative of progressive synapse elimination in the ventrolateral area. To verify that regression of the axons in vivo paralleled the changes in spatial distribution of fEPSPs as observed in vitro, corticospinal axons were anterogradely labeled. Redistribution of the labeled terminals closely paralleled the fEPSP distribution, being present in the ventrolateral spinal cord at P7, decreased at P8, further deceased at P10, but unchanged at P11. Furthermore, double immunostaining for labeled terminals and synaptophysin observed under a confocal microscope suggests that corticospinal fibers at P7 possess presynaptic structures in the ventrolateral area as well as the dorsomedial area. These findings suggest that corticospinal synapses are widely formed in the spinal gray matter at P7, are rapidly eliminated from the ventrolateral side from P8 to P10, a time-course very similar to that observed in vitro, and are associated with axonal regression.
Sensory and Motor Effects of Etomidate Anesthesia
Authors: Engelmann Jacob , Bacelo Joao , van den Burg Erwin , Grant Kirsty
Published: November 2005

The effects of anesthesia with etomidate on the cellular mechanisms of sensory processing and sensorimotor coordination have been studied in the active electric sense of the mormyrid fish Gnathonemus petersii. Like many anesthetics, etomidate is known to potentiate GABAAreceptors, but little is known about the effects on sensory processing at the systems level. A better understanding is necessary for experimental studies of sensory processing, in particular regarding possible effects on the dynamic structure of excitatory and inhibitory receptive fields and to improve the knowledge of the mechanisms of anesthesia in general. Etomidate slowed the electromotor discharge rhythm, probably because of feedback inhibition at the premotor level, but did not alter the structure of the electromotor command. Sensory translation through primary afferents projecting to the cerebellum-like electrosensory lobe (ELL) was not changed. However, central interneurons and projection neurons were hyperpolarized under etomidate, and their spiking activity was reduced. Although the spatial extent and the center/surround organization of sensory receptive fields were not changed, initial excitatory responses were followed by prolonged inhibition. Corollary discharge input to ELL was maintained, and the temporal sequence of excitatory and inhibitory components of this descending signal remained intact. Later inhibitory corollary discharge responses were prolonged by several hundred milliseconds. The result was that excitatory reafferent sensory input was conserved with enhanced precision of timing, whereas background activity was greatly reduced. Anti-Hebbian synaptic plasticity evoked by association of sensory and corollary discharge input was still present under anesthesia, and differences compared with the nonanesthetized condition are discussed.
Processing of Kinetic Boundaries in Macaque V4
Authors: Mysore Santosh G. , Vogels Rufin , Raiguel Steve E. , Orban Guy A.
Published: November 2005

We used gratings and shapes defined by relative motion to study selectivity for static kinetic boundaries in macaque V4 neurons. Kinetic gratings were generated by random pixels moving in opposite directions in the neighboring bars, either parallel to the orientation of the boundary (parallel kinetic grating) or perpendicular to the boundary (orthogonal kinetic grating). Neurons were also tested with static, luminance defined gratings to establish cue invariance. In addition, we used eight shapes defined either by relative motion or by luminance contrast, as used previously to test cue invariance in the infero-temporal (IT) cortex. A sizeable fraction (10–20%) of the V4 neurons responded selectively to kinetic patterns. Most neurons selective for kinetic contours had receptive fields (RFs) within the central 10° of the visual field. Neurons selective for the orientation of kinetic gratings were defined as having similar orientation preferences for the two types of kinetic gratings, and the vast majority of these neurons also retained the same orientation preference for luminance defined gratings. Also, kinetic shape selective neurons had similar shape preferences when the shape was defined by relative motion or by luminance contrast, showing a cue-invariant form processing in V4. Although shape selectivity was weaker in V4 than what has been reported in the IT cortex, cue invariance was similar in the two areas, suggesting that invariance for luminance and motion cues of IT originates in V4. The neurons selective for kinetic patterns tended to be clustered within dorsal V4.
Coding of Stimulus Frequency by Latency in Thalamic Networks Through the Interplay of GABAB-Mediated Feedback and Stimulus Shape
Authors: Golomb David , Ahissar Ehud , Kleinfeld David
Published: November 2005

A temporal sensory code occurs in posterior medial (POm) thalamus of the rat vibrissa system, where the latency for the spike rate to peak is observed to increase with increasing frequency of stimulation between 2 and 11 Hz. In contrast, the latency of the spike rate in the ventroposterior medial (VPm) thalamus is constant in this frequency range. We consider the hypothesis that two factors are essential for latency coding in the POm. The first is GABAB-mediated feedback inhibition from the reticular thalamic (Rt) nucleus, which provides delayed and prolonged input to thalamic structures. The second is sensory input that leads to an accelerating spike rate in brain stem nuclei. Essential aspects of the experimental observations are replicated by the analytical solution of a rate-based model with a minimal architecture that includes only the POm and Rt nuclei, i.e., an increase in stimulus frequency will increase the level of inhibitory output from Rt thalamus and lead to a longer latency in the activation of POm thalamus. This architecture, however, admits period-doubling at high levels of GABAB-mediated conductance. A full architecture that incorporates the VPm nucleus suppresses period-doubling. A clear match between the experimentally measured spike rates and the numerically calculated rates for the full model occurs when VPm thalamus receives stronger brain stem input and weaker GABAB-mediated inhibition than POm thalamus. Our analysis leads to the prediction that the latency code will disappear if GABAB-mediated transmission is blocked in POm thalamus or if the onset of sensory input is too abrupt. We suggest that GABAB-mediated inhibition is a substrate of temporal coding in normal brain function.
Identification of an Aberrantly Spliced Form of HDMX in Human Tumors: A New Mechanism for HDM2 Stabilization
Authors: Giglio Simona , Mancini Francesca , Gentiletti Francesca , Sparaco Giorgia , Felicioni Lara , Barassi Fabio , Martella Carla , Prodosmo Andrea , Iacovelli Stefano , Buttitta Fiamma , Farsetti Antonella , Soddu Silvia , Marchetti Antonio , Sacchi Ada , Pontecorvi Alfredo , Moretti Fabiola
Published: November 2005

The HDMX protein is closely related to HDM2 with which it shares different structural domains, particularly the p53 binding domain and the ring finger domain, where the two HDM proteins interact. Several oncogenic forms derived from splicing of HDM2 have been described in cancer. This work aimed at investigating whether analogous forms of HDMX exist in human tumors. Here, we report the characterization of an aberrantly spliced form of HDMX, HDMX211, isolated from the thyroid tumor cell line, ARO. HDMX211 binds and stabilizes the HDM2 protein. Although it lacks the p53 binding domain, HDMX211 also stabilizes p53 by counteracting its degradation by HDM2. However, the resulting p53 is transcriptionally inactive and increasingly associated to its inhibitor HDM2. Expression of HDMX211 strongly enhances the colony-forming ability of human cells in the presence or absence of wild-type p53. Conversely, depletion of HDMX211 by small interfering RNA significantly reduces the growth of ARO cells and increases their sensitivity to chemotherapy. Screening of lung cancer biopsies shows the presence of HDMX211 in samples that overexpress HDM2 protein, supporting a pathologic role for this new protein. This is the first evidence of a variant form of HDMX that has oncogenic potential independently of p53. HDMX211 reveals a new mechanism for overexpression of the oncoprotein HDM2. Most interestingly, it outlines a possible molecular explanation for a yet unclarified tumor phenotype, characterized by simultaneous overexpression of HDM2 and wild-type p53.
Proteolysis of CCN1 by Plasmin: Functional Implications
Authors: Pendurthi Usha R. , Tran Tien T. , Post Marina , Rao L. Vijaya Mohan
Published: November 2005

Plasmin is shown to play a crucial role in many pathophysiologic processes primarily through its ability to degrade extracellular matrix (ECM) and/or mobilizing growth factors that are sequestered in the ECM. Cysteine-rich 61 (CCN1) is a matricellular protein of which expression is up-regulated in cancer and various vascular diseases. The present study was undertaken to investigate whether plasmin liberates CCN1 from the ECM and whether the released growth factor modulates endothelial cell migration. Treatment of breast carcinoma cells (MDA-MB-231) with plasmin released a truncated form of CCN1 (28 kDa) into the overlying medium. Experiments with recombinant CCN1 confirmed that plasmin effectively cleaves CCN1. Thrombin and other clotting/fibrinolytic proteases are ineffective in cleaving CCN1. Further studies revealed that the conditioned medium of plasmin-treated carcinoma cells supports endothelial cell migration and that antibodies specific to CCN1 blocked this enhancing effect. These data were the first to show that plasmin can liberate a pluripotent matrix signaling protein, CCN1, from the ECM. Because both CCN1 and the components of the plasmin generation system are present in tumor cells and a variety of other cells, the proteolysis of CCN1 by plasmin may play a role in many pathophysiologic processes, including tumor cell–mediated angiogenesis.
A Role for Polymerase η in the Cellular Tolerance to Cisplatin-Induced Damage
Authors: Albertella Mark R. , Green Catherine M. , Lehmann Alan R. , O'Connor Mark J.
Published: November 2005

Mutation of the POLH gene encoding DNA polymerase η (pol η) causes the UV-sensitivity syndrome xeroderma pigmentosum-variant (XP-V) which is linked to the ability of pol η to accurately bypass UV-induced cyclobutane pyrimidine dimers during a process termed translesion synthesis. Pol η can also bypass other DNA damage adducts in vitro, including cisplatin-induced intrastrand adducts, although the physiological relevance of this is unknown. Here, we show that independent XP-V cell lines are dramatically more sensitive to cisplatin than the same cells complemented with functional pol η. Similar results were obtained with the chemotherapeutic agents, carboplatin and oxaliplatin, thus revealing a general requirement for pol η expression in providing tolerance to these platinum-based drugs. The level of sensitization observed was comparable to that of XP-A cells deficient in nucleotide excision repair, a recognized and important mechanism for repair of cisplatin adducts. However, unlike in XP-A cells, the absence of pol η expression resulted in a reduced ability to overcome cisplatin-induced S phase arrest, suggesting that pol η is involved in translesion synthesis past these replication-blocking adducts. Subcellular localization studies also highlighted an accumulation of nuclei with pol η foci that correlated with the formation of monoubiquitinated proliferating cell nuclear antigen following treatment with cisplatin, reminiscent of the response to UV irradiation and further indicating a role for pol η in dealing with cisplatin-induced damage. Together, these data show that pol η represents an important determinant of cellular responses to cisplatin, which could have implications for acquired or intrinsic resistance to this key chemotherapeutic agent.
Tocopherol-Associated Protein Suppresses Prostate Cancer Cell Growth by Inhibition of the Phosphoinositide 3-Kinase Pathway
Authors: Ni Jing , Wen Xingqiao , Yao Jorge , Chang Hong-Chiang , Yin Yi , Zhang Min , Xie Shaozhen , Chen Ming , Simons Brenna , Chang Philip , di Sant'Agnese Anthony , Messing Edward M. , Yeh Shuyuan
Published: November 2005

Epidemiologic studies suggested that vitamin E has a protective effect against prostate cancer. We showed here that tocopherol-associated protein (TAP), a vitamin E–binding protein, promoted vitamin E uptake and facilitated vitamin E antiproliferation effect in prostate cancer cells. Interestingly, without vitamin E treatment, overexpression of TAP in prostate cancer cells significantly suppressed cell growth; knockdown of endogenous TAP by TAP small interfering RNA (siRNA) in nonmalignant prostate HPr-1 cells increased cell growth. Further mechanism dissection studies suggested that the tumor suppressor function of TAP was via down-regulation of phosphoinositide 3-kinase (PI3K)/Akt signaling, but not by modulating cell cycle arrest or androgen receptor signaling. Immunoprecipitation results indicated that TAP inhibited the interaction of PI3K subunits, p110 with p85, and subsequently reduced Akt activity. Constitutively active Akt could negate the TAP-suppressive activity on prostate cancer cell growth. Moreover, stable transfection of TAP in LNCaP cells suppressed LNCaP tumor incidence and growth rate in nude mice. Furthermore, TAP mRNA and protein expression levels were significantly down-regulated in human prostate cancer tissue samples compared with benign prostate tissues as measured by reverse transcription-PCR, in situ hybridization, and immunohistochemistry. Together, our data suggest that TAP not only mediates vitamin E absorption to facilitate vitamin E antiproliferation effect in prostate cancer cells, but also functions like a tumor suppressor gene to control cancer cell viability through a non–vitamin E manner. Therefore, TAP may represent a new prognostic marker for prostate cancer progression.
HER2/neu-Induced Mammary Tumorigenesis and Angiogenesis Are Reduced in Cyclooxygenase-2 Knockout Mice
Authors: Howe Louise R. , Chang Sung-Hee , Tolle Kelly C. , Dillon Rachelle , Young Lawrence J.T. , Cardiff Robert D. , Newman Robert A. , Yang Peiying , Thaler Howard T. , Muller William J. , Hudis Clifford , Brown Anthony M.C. , Hla Timothy , Subbaramaiah Kotha , Dannenberg Andrew J.
Published: November 2005

The inducible prostaglandin synthase cyclooxygenase-2 (Cox-2) is overexpressed in ∼40% of human breast cancers and at higher frequencies in preinvasive ductal carcinoma in situ (DCIS). Cox-2 expression is particularly associated with overexpression of human epidermal growth factor receptor 2 (HER2/neu). To definitively interrogate the role of Cox-2 in mammary neoplasia, we have used a genetic approach, crossing Cox-2-deficient mice with a HER2/neu transgenic strain, MMTV/NDL. At 20 weeks of age, mammary glands from virgin MMTV/NDL females contained multiple focal tumors, or mammary intraepithelial neoplasias, which histologically resembled human DCIS. Mammary tumor multiplicity and prostaglandin E2 (PGE2) levels were significantly decreased in Cox-2 heterozygous and knockout animals relative to Cox-2 wild-type controls. Notably, the proportion of larger tumors was decreased in Cox-2-deficient mice. HER2/neu-induced mammary hyperplasia was also substantially reduced in Cox-2 null mice. Additionally, mammary glands from Cox-2 knockout mice exhibited a striking reduction in vascularization, and expression of proangiogenic genes was correspondingly reduced. Decreased vascularization was observed both in dysplastic and normal-appearing regions of Cox-2-null mammary glands. Our data provide the first genetic evidence that Cox-2 contributes to HER2/neu-induced mammary tumorigenesis. This finding may help to explain the reduced risk of breast cancer associated with regular use of nonsteroidal anti-inflammatory drugs.
Mutations of C-RAF Are Rare in Human Cancer because C-RAF Has a Low Basal Kinase Activity Compared with B-RAF
Authors: Emuss Victoria , Garnett Mathew , Mason Clive ,   The Cancer Genome Project, Marais Richard
Published: November 2005

The protein kinase B-RAF is mutated in ∼8% of human cancers. Here we show that presumptive mutants of the closely related kinase, C-RAF, were detected in only 4 of 545 (0.7%) cancer cell lines. The activity of two of the mutated proteins is not significantly different from that of wild-type C-RAF and these variants may represent rare human polymorphisms. The basal and B-RAF–stimulated kinase activities of a third variant are unaltered but its activation by RAS is significantly reduced, suggesting that it may act in a dominant-negative manner to modulate pathway signaling. The fourth variant has elevated basal kinase activity and is hypersensitive to activation by RAS but does not transform mammalian cells. Furthermore, when we introduce the equivalent of the most common cancer mutation in B-RAF (V600E) into C-RAF, it only has a weak effect on kinase activity and does not convert C-RAF into an oncogene. This lack of activation occurs because C-RAF lacks a constitutive charge within a motif in the kinase domain called the N-region. This fundamental difference in RAF isoform regulation explains why B-RAF is frequently mutated in cancer whereas C-RAF mutations are rare.
Asbestos-Induced Lung Inflammation and Epithelial Cell Proliferation Are Altered in Myeloperoxidase-Null Mice
Authors: Haegens Astrid , van der Vliet Albert , Butnor Kelly J. , Heintz Nicholas , Taatjes Douglas , Hemenway David , Vacek Pamela , Freeman Bruce A. , Hazen Stanley L. , Brennan Marie Luise , Mossman Brooke T.
Published: November 2005

Asbestos fibers are carcinogens causing oxidative stress and inflammation, but the sources and ramifications of oxidant production by asbestos are poorly understood. Here, we show that inhaled chrysotile asbestos fibers cause increased myeloperoxidase activity in bronchoalveolar lavage fluids (BALF) and myeloperoxidase immunoreactivity in epithelial cells lining distal bronchioles and alveolar ducts, sites of initial lung deposition of asbestos fibers. In comparison with sham mice, asbestos-exposed myeloperoxidase-null (MPO−/−) and normal (MPO+/+) mice exhibited comparable increases in polymorphonuclear leukocytes, predominately neutrophils, in BALF after 9 days of asbestos inhalation. Differential cell counts on BALF revealed decreased proportions of macrophages and increased lymphocytes in all mice exposed to asbestos, but numbers were decreased overall in asbestos-exposed myeloperoxidase-null versus normal mice. Asbestos-associated lung inflammation in myeloperoxidase-null mice was reduced (P ≤ 0.05) in comparison with normal asbestos-exposed mice at 9 days. Decreased lung inflammation in asbestos-exposed myeloperoxidase-null mice at 9 days was accompanied by increases (P ≤ 0.05) in Ki-67- and cyclin D1-positive immunoreactive cells, markers of cell cycle reentry, in the distal bronchiolar epithelium. Asbestos-induced epithelial cell proliferation in myeloperoxidase-null mice at 30 days was comparable to that found at 9 days. In contrast, inflammation and epithelial cell proliferation in asbestos-exposed normal mice increased over time. These results support the hypothesis that myeloperoxidase status modulates early asbestos-induced oxidative stress, epithelial cell proliferation, and inflammation.
Bombesin Regulates Cyclin D1 Expression through the Early Growth Response Protein Egr-1 in Prostate Cancer Cells
Authors: Xiao Dongmei , Chinnappan Dharmaraj , Pestell Richard , Albanese Christopher , Weber Horst Christian
Published: November 2005

Our previous studies indicate that the activation of mitogen-activated protein kinase (MAPK) pathway is involved in bombesin-induced cell proliferation in prostate cancer cells. Cyclin D1 is a critical regulator involved in cell cycle progression through the G1 phase into the S phase, thereby contributing to cell proliferation. Mostly, mitogen-stimulated expression of cyclin D1 is attributed to the extracellular signal-regulated kinase (ERK) activation. Here, we found that bombesin induced human cyclin D1 expression on both mRNA and protein levels in DU-145 prostate cancer cells. Mutational analyses showed that bombesin-enhanced cyclin D1 transcription required the binding of nuclear proteins to the −143 to −105 region of the human cyclin D1 promoter, which contains binding sites for transcription factors Sp-1 and early growth response protein (Egr-1). Do novo protein synthesis was requisite for bombesin-induced cyclin D1 expression. Further studies showed Egr-1 was induced upon bombesin stimulation. The induction of Egr-1 expression and its binding to the cyclin D1 promoter were essential for bombesin-enhanced cyclin D1 transcription. Inhibition of MAPK pathway with either the MEK1 inhibitor PD98059 or a dominant-negative Ras mutant, RasN17, abolished bombesin-induced cyclin D1 activation. Taken together, bombesin-induced cyclin D1 expression in prostate cancer cells is mediated by Egr-1 activation and the interaction of Egr-1 with the Egr-1/Sp1 motif of the cyclin D1 promoter through the activation of MAPK pathway. These findings represent a novel mechanism of bombesin-dependent stimulation of mitogenesis by regulating directly the cell cycle in prostate cancer.
Cerebrospinal Fluid Proteomic Analysis Reveals Dysregulation of Methionine Aminopeptidase-2 Expression in Human and Mouse Neurofibromatosis 1–Associated Glioma
Authors: Dasgupta Biplab , Yi Yijun , Hegedus Balazs , Weber Jason D. , Gutmann David H.
Published: November 2005

Individuals affected with the neurofibromatosis 1 (NF1) tumor predisposition syndrome are prone to the development of multiple nervous system tumors, including optic pathway gliomas (OPG). The NF1 tumor suppressor gene product, neurofibromin, functions as a Ras GTPase-activating protein, and has been proposed to regulate cell growth by inhibiting Ras activity. Recent studies from our laboratory have shown that neurofibromin also regulates the mammalian target of rapamycin activity in a Ras-dependent fashion, and that the rapamycin-mediated mammalian target of rapamycin inhibition ameliorates the Nf1−/− astrocyte growth advantage. Moreover, Nf1-deficient astrocytes exhibit increased protein translation. As part of a larger effort to identify protein markers for NF1-associated astrocytomas that could be exploited for therapeutic drug design, we did an objective proteomic analysis of the cerebrospinal fluid from genetically engineered Nf1 mice with optic glioma. One of the proteins found to be increased in the cerebrospinal fluid of OPG-bearing mice was the eukaryotic initiation factor-2α binding protein, methionine aminopeptidase 2 (MetAP2). In this study, we show that Nf1 mouse OPGs and NF1-associated human astrocytic tumors, but not sporadic pilocytic or other low-grade astrocytomas, specifically expressed high levels of MetAP2. In addition, we show that Nf1-deficient astrocytes overexpress MetAP2 in vitro and in vivo, and that treatment with the MetAP2 inhibitor fumagillin significantly reduces Nf1−/− astrocyte proliferation in vitro. These observations suggest that MetAP2 is regulated by neurofibromin, and that MetAP2 inhibitors could be potentially employed to treat NF1-associated tumor proliferation.
Unexpected Roles for pRb in Mouse Skin Carcinogenesis
Authors: Ruiz Sergio , Santos Mirentxu , Lara M. Fernanda , Segrelles Carmen , Ballestín Claudio , Paramio Jesús M.
Published: November 2005

The mouse skin carcinogenesis represents one of the best models for the understanding of malignant transformation, including the multistage nature of tumor development. The retinoblastoma gene product (pRb) plays a critical role in cell cycle regulation, differentiation, and inhibition of oncogenic transformation. In epidermis, Rb−/− deletion leads to proliferation and differentiation defects. Numerous evidences showed the involvement of the retinoblastoma pathway in this model. However, the actual role of pRb is still unknown. To study the possible involvement of pRb in keratinocyte malignant transformation, we have carried out two-stage chemical skin carcinogenesis on RbF19/F19 (thereafter Rb+/+) and RbF19/F19;K14Cre (thereafter Rb−/−) animals. Unexpectedly, we found that Rb−/− mice developed fewer and smaller papillomas than the Rb+/+ counterparts. Moreover, the small size of the pRb-deficient tumors is associated with an increase in the apoptotic index. Despite this, pRb-deficient tumors display an increased conversion rate to squamous cell carcinomas. Biochemical analyses revealed that these characteristics correlate with the differential expression and activity of different pathways, including E2F/p19arf/p53, PTEN/Akt, c-jun NH2-terminal kinase/p38, and nuclear factor-κB. Collectively, our findings show unexpected and hitherto nondescribed roles of pRb during the process of epidermal carcinogenesis.
Doxorubicin Induces Apoptosis in Germ Line Stem Cells in the Immature Rat Testis and Amifostine Cannot Protect against This Cytotoxicity
Authors: Hou Mi , Chrysis Dionisios , Nurmio Mirja , Parvinen Martti , Eksborg Staffan , Söder Olle , Jahnukainen Kirsi
Published: November 2005

The underlying primary damage to the seminiferous epithelium caused by chemotherapeutic regimens at childhood is largely unknown. The present investigation was designed to identify acute cytotoxic events in the testis caused by a single dose of doxorubicin. Male rats at 6, 16, and 24 days of age were injected with doxorubicin (3 mg/kg, i.p.) or vehicle (saline) alone and 24 and 48 hours later, the germ cell types and apoptotic cells in the seminiferous epithelium were examined.

As indicated by microscopy and terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling staining, an 8-fold increase in the number of apoptotic germ cells in the testes of 6-day-old rats was observed 48 hours after doxorubicin treatment. Spermatogonia migrating to the basement membrane were the primary cell type undergoing this induced apoptosis. A single dose of amifostine (200 mg/kg) administered i.p. 15 minutes before injection of doxorubicin provided no protection against this enhanced apoptosis. Under the same conditions, testicular levels of p53 and activated caspase 8 were elevated, whereas the level of murine double minute-2 was lowered. In contrast, doxorubicin treatment did not result in any significant change in the physiologic, stage-specific germ cell apoptosis occurring in the testes of 16- and 24-day-old rats. These observations suggest that the initiation phase of spermatogenesis is highly sensitive to doxorubicin-induced apoptosis. Gonocytes and early spermatogonia are the cell types that are vulnerable to this p53-trigged apoptosis, which results in a decrease in the size of the pool of germ-line stem cells. Amifostine fails to protect the germ cells against this cytotoxic insult.