bluuebunny/crossref_metadata_2025 huggingface dataset:
Sex differences in the alterations of Na+,K+‐ATPase following ischaemia–reperfusion injury in the rat kidney
Authors: Fekete Andrea , Vannay Ádám , Vér Ágota , Vásárhelyi Barna , Müller Veronika , Ouyang Nengtai , Reusz György , Tulassay Tivadar , Szabó Attila J.
Published: December 2003

Postischaemic acute renal failure (ARF) is influenced by sex. Na+,K+‐ATPase (NKA) plays a crucial role in the pathogenesis of postischaemic ARF. We tested the impact of sex on mRNA, protein expression, cellular distribution and enzyme activity of NKA following renal ischaemia–reperfusion (I‐R) injury. The left renal pedicle of uninephrectomized female (F) and male (M) Wistar rats was clamped for 55 min followed by 2 h (T2) and 16 h (T16) of reperfusion. Uninephrectomized, sham‐operated F and M rats served as controls (n= 6 per group). Blood urea nitrogen, serum creatinine and renal histology were evaluated to detect the severity of postischaemic ARF. mRNA expression of NKA α1 and β1 subunits were detected by RT‐PCR. The effect of I‐R on cellular distribution was compared by Triton X‐100 extraction. Cellular proteins were divided into Triton‐insoluble and Triton‐soluble fractions and assessed by Western blot. NKA enzyme activity was also determined. After the ischaemic insult blood urea nitrogen and serum creatinine were higher and renal histology showed more rapid progression in M versus  F (P < 0.05). mRNA expression of the NKA α1 subunit decreased in I‐R groups versus controls, but was higher in F versus M both in control and I‐R groups (P < 0.05). However, protein levels of the NKA α1 subunit in total tissue homogenate did not differ in controls, but were higher in F versus M in I‐R groups (P < 0.05). Triton X‐100 extractability was lower in F versus M at T16 (P < 0.05). NKA enzyme activity was the same in controls, but was higher in F versus M in I‐R groups (T2: 14.9 ± 2.3 versus 9.15 ± 2.21 U) (T16: 11.7 ± 4.1 versus 5.65 ± 2.3 U; P < 0.05). mRNA and protein expression of the NKA β1 subunit did not differ between F and M in any of the protocol. We concluded that NKA is more protected from the detrimental effects of postischaemic injury in females. Higher mRNA and protein expression of the NKA α1 subunit and higher enzyme activity might be additional contributing factors to the improved postischaemic renal function of female rats.
Actions of noradrenaline on substantia gelatinosa neurones in the rat spinal cord revealed by in vivo patch recording
Authors: Sonohata Motoki , Furue Hidemasa , Katafuchi Toshihiko , Yasaka Toshiharu , Doi Atsushi , Kumamoto Eiichi , Yoshimura Megumu
Published: December 2003

To elucidate the mechanisms of antinociception mediated by the descending noradrenergic pathway in the spinal cord, the effects of noradrenaline (NA) on noxious synaptic responses of substantia gelatinosa (SG) neurones, and postsynaptic actions of NA were investigated in rats using an in vivo whole‐cell patch‐clamp technique. Under urethane anaesthesia, the rat was fixed in a stereotaxic apparatus after the lumbar spinal cord was exposed. In the current‐clamp mode, pinch stimuli applied to the ipsilateral hindlimb elicited a barrage of EPSPs, some of which initiated an action potential. Perfusion with NA onto the surface of the spinal cord hyperpolarized the membrane (5.0–9.5 mV) and suppressed the action potentials. In the voltage‐clamp mode (VH, −70 mV), the application of NA produced an outward current that was blocked by Cs+ and GDP‐β‐S added to the pipette solution and reduced the amplitude of EPSCs evoked by noxious stimuli. Under the blockade of postsynaptic actions of NA, a reduction of the evoked and spontaneous EPSCs of SG neurones was still observed, thus suggesting both pre‐ and postsynaptic actions of NA. The NA‐induced outward currents showed a clear dose dependency (EC50, 20 μm), and the reversal potential was −88 mV. The outward current was mimicked by an α2‐adrenoceptor agonist, clonidine, and suppressed by an α2‐adrenoceptor antagonist, yohimbine, but not by α1‐ and β‐antagonists. These findings suggest that NA acts on presynaptic sites to reduce noxious stimuli‐induced EPSCs, and on postsynaptic SG neurones to induce an outward current by G‐protein‐mediated activation of K+ channels through α2‐adrenoceptors, thereby producing an antinociceptive effect.
Activation and integration of bilateral GABA‐mediated synaptic inputs in neonatal rat sympathetic preganglionic neurones in vitro
Authors: Whyment Andrew D. , Wilson Jennifer M. M. , Renaud Leo P. , Spanswick David
Published: December 2003

The role of GABA receptors in synaptic transmission to neonatal rat sympathetic preganglionic neurones (SPNs) was investigated utilizing whole‐cell patch clamp recording techniques in longitudinal and transverse spinal cord slice preparations. In the presence of glutamate receptor antagonists (NBQX, 5 μm and D‐APV, 10 μm), electrical stimulation of the ipsilateral or contralateral lateral funiculi (iLF and cLF, respectively) revealed monosynaptic inhibitory postsynaptic potentials (IPSPs) in 75% and 65% of SPNs, respectively. IPSPs were sensitive to bicuculline (10 μm) in all neurones tested and reversed polarity around −55 mV, the latter indicating mediation via chloride conductances. In three neurones IPSPs evoked by stimulation of the iLF (n= 1) or cLF (n= 2) were partly sensitive to strychnine (2 μm). The expression of postsynaptic GABAA and GABAB receptors were confirmed by the sensitivity of SPNs to agonists, GABA (2 mm), muscimol (10–100 μm) or baclofen (10–100 μm), in the presence of TTX, each of which produced membrane hyperpolarization in all SPNs tested. Muscimol‐induced responses were sensitive to bicuculline (1–10 μm) and SR95531 (10 μm) and baclofen‐induced responses were sensitive to 2‐hydroxy‐saclofen (100–200 μm) and CGP55845 (200 nm). The GABAC receptor agonist CACA (200 μm) was without significant effect on SPNs. These results suggest that SPNs possess postsynaptic GABAA and GABAB receptors and that subsets of SPNs receive bilateral GABAergic inputs which activate GABAA receptors, coupled to a chloride conductance. At resting or holding potentials close to threshold either single or bursts (10–100 Hz) of IPSPs gave rise to a rebound excitation and action potential firing at the termination of the burst. This effect was mimicked by injection of small (10–20 pA) rectangular‐wave current pulses, which revealed a time‐dependent, Cs+‐sensitive inward rectification and rebound excitation at the termination of the response to current injection. Synaptic activation of a rebound excitation mediated by a time‐dependent inward rectification expressed intrinsically by SPNs may provide a novel mechanism enabling SPNs to be entrained to rhythms driven from the brainstem or higher centres.
Maternal melatonin selectively inhibits cortisol production in the primate fetal adrenal gland
Authors: Torres‐Farfan Claudia , Richter Hans G. , Germain Alfredo M. , Valenzuela Guillermo J. , Campino Carmen , Rojas‐García Pedro , Forcelledo María Luisa , Torrealba Fernando , Serón‐Ferré María
Published: December 2003

We tested the hypothesis that in primates, maternal melatonin restrains fetal and newborn adrenal cortisol production. A functional G‐protein‐coupled MT1 membrane‐bound melatonin receptor was detected in 90% gestation capuchin monkey fetal adrenals by (a) 2‐[125I] iodomelatonin binding (Kd, 75.7 ± 6.9 pm; Bmax, 2.6 ± 0.4 fmol (mg protein)−1), (b) cDNA identification, and (c) melatonin inhibition of adrenocorticotrophic hormone (ACTH)‐ and corticotrophin‐releasing hormone (CRH)‐stimulated cortisol but not of dehydroepiandrosterone sulphate (DHAS) production in vitro. Melatonin also inhibited ACTH‐induced 3β‐hydroxysteroid dehydrogenase mRNA expression. To assess the physiological relevance of these findings, we next studied the effect of chronic maternal melatonin suppression (induced by exposure to constant light during the last third of gestation) on maternal plasma oestradiol during gestation and on plasma cortisol concentration in the 4‐ to 6‐day‐old newborn. Constant light suppressed maternal melatonin without affecting maternal plasma oestradiol concentration, consistent with no effect on fetal DHAS, the precursor of maternal oestradiol. However, newborns from mothers under constant light condition had twice as much plasma cortisol as newborns from mothers maintained under a normal light–dark schedule. Newborns from mothers exposed to chronic constant light and daily melatonin replacement had normal plasma cortisol concentration. Our results support a role of maternal melatonin in fetal and neonatal primate cortisol regulation.
Cholecystokinin activates CCKB receptors to excite cells and depress EPSCs in the rat rostral nucleus accumbens in vitro
Authors: Kombian Samuel B. , Ananthalakshmi Kethireddy V. V. , Parvathy Subramanian S. , Matowe Wandikayi C.
Published: December 2003

The peptide cholecystokinin (CCK) is abundant in the rat nucleus accumbens (NAc). Although it is colocalized with dopamine (DA) in afferent terminals in this region, neurochemical and behavioural reports are equally divided as to whether CCK enhances or diminishes DA's actions in this nucleus. To better understand the role of this peptide in the physiology of the NAc, we examined the effects of CCK on excitatory synaptic transmission and tested whether these are dependent on DA and/or other neuromodulators. Using whole‐cell recording in rat forebrain slices containing the NAc, we show that sulphated CCK octapeptide (CCK‐8S), the endogenously active neuropeptide, consistently depolarized cells and depressed evoked excitatory postsynaptic currents (EPSCs) in the rostral NAc. It caused a reversible, dose‐dependent decrease in evoked EPSC amplitude that was accompanied by an increase in the decay constant of the EPSC but with no apparent change in paired pulse ratio. It was mimicked by unsulphated CCK‐8 (CCK‐8US), a CCKB receptor‐selective agonist, and blocked by LY225910, a CCKB receptor‐selective antagonist. Both CCK‐8S and CCK‐8US induced an inward current with a reversal potential around −90 mV that was accompanied by an increase in input resistance and action potential firing. The CCK‐8S‐induced EPSC depression was slightly reduced in the presence of SCH23390 but not in the presence of sulpiride or 8‐cyclopentyltheophylline. By contrast, it was completely blocked by CGP55845, a potent GABAB receptor‐selective antagonist. These results indicate that CCK excites NAc cells directly while depressing evoked EPSCs indirectly, mainly through the release of GABA.
Cannabinoid‐induced presynaptic inhibition at the primary afferent trigeminal synapse of juvenile rat brainstem slices
Authors: Liang Ying‐Ching , Huang Chiung‐Chun , Hsu Kuei‐Sen , Takahashi Tomoyuki
Published: December 2003

Systemic or intraventricular administration of cannabinoids causes analgesic effects, but relatively little is known for their cellular mechanism. Using brainstem slices with the mandibular nerve attached, we examined the effect of cannabinoids on glutamatergic transmission in superficial trigeminal caudal nucleus of juvenile rats. The exogenous cannabinoid receptor agonist WIN 55,212‐2 (WIN), as well as the endogenous agonist anandamide, hyperpolarized trigeminal caudal neurones and depressed the amplitude of excitatory postsynaptic potentials (EPSPs) or currents (EPSCs) monosynaptically evoked by stimulating mandibular nerves in a concentration‐dependent manner. The inhibitory action of WIN was blocked or fully reversed by the CB1 receptor antagonist SR 141716A. WIN had no effect on the amplitude of miniature excitatory postsynaptic currents (mEPSCs) recorded in the presence of tetrodotoxin or cadmium. The inhibitory effect of WIN on EPSCs was greater for those with longer synaptic latency, suggesting that cannabinoids have a stronger effect on C‐fibre EPSPs than on Aδ‐fibre EPSPs. Ba2+ (100 μm) blocked the hyperpolarizing effect of cannabinoids, but did not affect their inhibitory effect on EPSPs. The N‐type Ca2+ channel blocker ω‐conotoxin GVIA (ω‐CgTX) occluded the WIN‐mediated presynaptic inhibition, whereas the P/Q‐type Ca2+ channel blocker ω‐agatoxin TK (ω‐Aga) had no effect. These results suggest that cannabinoids preferentially activate CB1 receptors at the nerve terminal of small‐diameter primary afferent fibres. Upon activation, CB1 receptors may selectively inhibit presynaptic N‐type Ca2+ channels and exocytotic release machinery, thereby attenuating the transmitter release at the trigeminal nociceptive synapses.
Attenuated outward potassium currents in carotid body glomus cells of heart failure rabbit: involvement of nitric oxide
Authors: Li Yu‐Long , Sun Shu‐Yu , Overholt Jeffery L. , Prabhakar Nanduri R. , Rozanski George J. , Zucker Irving H. , Schultz Harold D.
Published: December 2003

It has been shown that peripheral chemoreceptor sensitivity is enhanced in both clinical and experimental heart failure (HF) and that impairment of nitric oxide (NO) production contributes to this enhancement. In order to understand the cellular mechanisms associated with the alterations of chemoreceptor function and the actions of NO in the carotid body (CB), we compared the outward K+ currents (IK) of glomus cells in sham rabbits with that in HF rabbits and monitored the effects of NO on these currents. Ik was measured in glomus cells using conventional and perforated whole‐cell configurations. IK was attenuated in glomus cells of HF rabbits, and their resting membrane potentials (−34.7 ± 1.0 mV) were depolarized as compared with those in sham rabbits (−47.2 ± 1.9 mV). The selective Ca2+‐dependent K+ channel (KCa) blocker iberiotoxin (IbTx, 100 nm) reduced IK in glomus cells from sham rabbits, but had no effect on IK from HF rabbits. In perforated whole‐cell mode, the NO donor SNAP (100 μm) increased IK in glomus cells from HF rabbits to a greater extent than that in sham rabbits (P < 0.01), and IbTx inhibited the effects of SNAP. However, in conventional whole‐cell mode, SNAP had no effect. Nω‐nitro‐L‐arginine (L‐NNA, NO synthase inhibitor) decreased Ik in sham rabbits but not in HF rabbits. The guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one (ODQ) inhibited the effect of SNAP on Ik. These results demonstrate that IK is reduced in CB glomus cells from HF rabbits. This effect is due mainly to the suppression of KCa channel activity caused by decreased availability of NO. In addition, intracellular cGMP is necessary for the KCa channel modulation by NO.
Neonatal stress alters adult breathing
Authors: McCrimmon Donald R. , Alheid George F.
Published: December 2003

The epigenetic effect of deprivation and stress on brain development is one of the most enduring topics in neurobiology and one that arguably has had the most far reaching influences on modern societies. Persistent deficits in perception, cognition, and social development are attributed to maternal separation and/or environmental deprivation of human infants. Given the retrospective nature of these human studies, the animal experiments they have inspired are particularly significant insofar as objective changes in physiology, neuronal structure and brain size follow manipulations of the perinatal sensory environment.
Extracellular K+ accumulation: a physiological framework for fatigue during intense exercise
Authors: Bickham Dale C.
Published: December 2003

What are the causes of fatigue? Elevated lactate or lowered pH or failure of calcium release, I hear you say. Well yes, but under what conditions? The work of Nielsen et al. (2004) in this issue of The Journal of Physiology provides experimental evidence which sheds light on the mechanism of fatigue development during intense exercise.
Random sampling of an alternating current source: A tool for teaching probabilistic observations
Authors: Arvind  , Chandi P. S. , Singh R. C. , Indumathi D. , Shankar R.
Published: December 2003

We describe an undergraduate-level experiment that demonstrates the role of probabilistic observations in physics. A capacitor and a dc voltmeter are used to randomly sample an ac voltage. The resulting probability distribution is analyzed to extract information about the source. Different characteristic probability distributions arising from various ac waveforms are calculated and experimentally measured. We show how limited information about the waveform can be extracted from the voltage probability distribution, which is used to reconstruct the waveform for the special case of a sinusoidal waveform. The results are also compared with a simulated data sample. The experiment can be used as a pedagogical tool to teach probabilistic measurements and their manipulations.
Isotope/element fractionation during surface adsorption
Authors: Seneviratne Gamini , Nanayakkara Asiri
Published: December 2003

The adsorption of ions onto mineral surfaces accompanies isotope/element fractionation in planets and asteroids. A model based on simple classical physics is presented to predict these fractionations. The agreement between the experimentally observed isotope/element ratios and their predicted values is found to be excellent. This fractionation can be demonstrated experimentally in advanced physics laboratories using macroscopic particles. The success of the model shows students that even a very complex naturally occurring process can be explained quantitatively with simple physics.
Vacuum polarization and photon mass in inflation
Authors: Prokopec Tomislav , Woodard Richard
Published: December 2003

We give a pedagogical review of a mechanism through which long wavelength photons can become massive during inflation. Our account begins with a discussion of the period of exponentially rapid expansion known as inflation. We next describe how, when the universe is not expanding, quantum fluctuations in charged particle fields cause even empty space to behave as a polarizable medium. This is the routinely observed phenomenon of vacuum polarization. We show that the quantum fluctuations of low mass, scalar fields are enormously amplified during inflation. If one of these fields is charged, the vacuum polarization effect of flat space is strengthened to the point that long wavelength photons acquire mass. Our result for this mass is shown to agree with a simple model in which the massive photon electrodynamics of Proca emerges from applying the Hartree approximation to scalar quantum electrodynamics during inflation. A huge photon mass is not measured today because the original phase of inflation ended when the universe was only a tiny fraction of a second old. However, the zero-point energy left over from the epoch of large photon mass may have persisted during the post-inflationary universe as very weak, but cosmological-scale, magnetic fields. It has been suggested that these small seed fields were amplified by a dynamo mechanism to produce the micro-Gauss magnetic fields observed in galaxies and galactic clusters.
Use of electromagnetic scalar potentials in boundary value problems
Authors: Lahart Martin J.
Published: December 2003

Expressions for the electric and magnetic fields in terms of scalar potentials are derived, and boundary conditions that the scalar potentials obey at dielectric interfaces and at conducting surfaces are discussed. The relationship of the scalar potentials to source charges and currents is described. It is shown that scalar potentials can be defined that do not obey the wave equation, but the source charge and current configurations that can be associated with such potentials are more limited than those that do obey the wave equation. Examples are given of the use of electromagnetic scalar potentials in boundary value problems.
Simple applications of Noether’s first theorem in quantum mechanics and electromagnetism
Authors: Brown Harvey R. , Holland Peter
Published: December 2003

Internal global symmetries exist for the free nonrelativistic Schrödinger particle, whose associated Noether charges, space integrals of the wavefunction and wavefunction multiplied by the spatial coordinate, are exhibited. Analogous symmetries in classical electromagnetism also are demonstrated. These results illustrate a number of subtleties associated with Noether’s first theorem.
A Bell-type theorem without hidden variables
Authors: Stapp Henry P.
Published: December 2003

It is shown that no theory that satisfies certain premises can exclude faster-than-light influences. The premises include neither the existence of hidden variables nor counterfactual definiteness, nor any premise that effectively entails the general existence of outcomes of unperformed local measurements. All the premises are compatible with Copenhagen philosophy and the principles and predictions of relativistic quantum field theory. The present proof is contrasted with an earlier one with the same objective.
Students’ understanding of direct current resistive electrical circuits
Authors: Engelhardt Paula Vetter , Beichner Robert J.
Published: December 2003

Both high school and university students’ reasoning regarding direct current resistive electric circuits often differ from the accepted explanations. At present, there are no standard diagnostic tests on electric circuits. Two versions of a diagnostic instrument were developed, each consisting of 29 questions. The information provided by this test can provide instructors with a way of evaluating the progress and conceptual difficulties of their students. The analysis indicates that students, especially females, tend to hold multiple misconceptions, even after instruction. During interviews, the idea that the battery is a constant source of current was used most often in answering the questions. Students tended to focus on the current in solving problems and to confuse terms, often assigning the properties of current to voltage and/or resistance.
Virtual tunnels and green glass: The colors of common mirrors
Authors: Lee Raymond L. , Hernández-Andrés Javier
Published: December 2003

When a pair of common second-surface plane mirrors face each other, repeated mirror-to-mirror reflections form a virtual optical tunnel with some unusual properties. One property readily analyzed in a student experiment is that the color of objects becomes darker and greener the deeper we look into the mirror tunnel. This simple observation is both visually compelling and physically instructive: measuring and modeling a tunnel’s colors requires students to blend colorimetry and spectrophotometry with a knowledge of how complex refractive indices and the Fresnel equations predict reflectance spectra of composite materials.
The Thomas precession factor in spin–orbit interaction
Authors: Kroemer Herbert
Published: December 2003

The origin of the Thomas factor 1/2 in the spin–orbit Hamiltonian can be understood by considering the case of a classical electron moving in crossed electric and magnetic fields chosen such that the electric Coulomb force is balanced by the magnetic Lorentz force.
Extreme stiffness systems due to negative stiffness elements
Authors: Wang Y. C. , Lakes R. S.
Published: December 2003

When an elastic object is pressed, we expect it to resist by exerting a restoring force. A reversal of this force corresponds to negative stiffness. If we combine elements with positive and negative stiffness in a composite, it is possible to achieve stiffness greater than (or less than) that of any of the constituents. This behavior violates established bounds that tacitly assume that each phase has positive stiffness. Extreme composite behavior has been experimentally demonstrated in a lumped system using a buckled tube to achieve negative stiffness and in a composite material in the vicinity of a phase transformation of one of the constituents. In the context of a composite system, extreme refers to a physical property greater than either constituent. We consider a simple spring model with pre-load to achieve negative stiffness. When suitably tuned to balance positive and negative stiffness, the system shows a critical equilibrium point giving rise to extreme overall stiffness. A stability analysis of a viscous damped system containing negative stiffness springs reveals that the system is stable when tuned for high compliance, but metastable when tuned for high stiffness. The metastability of the extreme system is analogous to that of diamond. The frequency response of the viscous damped system shows that the overall stiffness increases with frequency and goes to infinity when one constituent has a suitable negative stiffness.
Neutrino physics: An update
Authors: Haxton Wick C. , Holstein Barry R.
Published: December 2003

We update our recent didactic survey of neutrino physics, including new results from the Sudbury Neutrino Observatory and Kamioka Liquid Scintillator Anti-Neutrino Detector experiments, and recent constraints from the Wilkinson Microwave Anisotropy Probe and other cosmological probes.
What good is the thermodynamic limit?
Authors: Styer Daniel F.
Published: December 2003

Statistical mechanics applies to large systems: technically, its results are exact only for infinitely large systems in “the thermodynamic limit.” The importance of this proviso is often minimized in undergraduate courses. This paper presents six paradoxes in statistical mechanics that can be resolved only by acknowledging the thermodynamic limit. For example, it demonstrates that the widely used microcanonical “thin phase space limit” must be taken after taking the thermodynamic limit.
Resource Letter: TA-1: Thermoacoustic engines and refrigerators
Authors: Garrett Steven L.
Published: December 2003

This Resource Letter provides an annotated guide to some of the literature pertaining to the understanding of thermoacoustic engines and refrigerators. These devices incorporate acoustical components and networks to produce mechanical power or to pump heat, or both, without the use of traditional mechanical contrivances such as pistons, linkages, and valves. To bring some order to this research and the variety of thermoacoustic engines and refrigerators produced over the past two decades, these devices also are classified as stack-based and regenerator-based. The background and motivation for this organizational structure is provided in the introduction.
The effect of distracters on student performance on the force concept inventory
Authors: Rebello N. Sanjay , Zollman Dean A.
Published: December 2003

We have compared students’ responses on four multiple-choice force concept inventory (FCI) questions with similar responses to equivalent open-ended questions. Our results indicate a good agreement between the percentages of correct responses in each of the two formats, indicating that distracters on the FCI do not adversely affect performance as measured by the number of correct answers. However, a significant percentage of the open-ended responses fall into categories that are not included in the FCI multiple choices. When these alternative categories were presented to the students as distracters in a revised multiple-choice format, a significant percentage of the students chose these alternative responses.
An Ion Balance for Ultra-High-Precision Atomic Mass Measurements
Authors: Rainville Simon , Thompson James K. , Pritchard David E.
Published: December 2003

We have developed the analog of a double-pan balance for determining the masses of single molecular ions from the ratio of their two cyclotron frequencies. By confining two different ions on the same magnetron orbit in a Penning trap, we balance out many sources of noise and error (such as fluctuations of the magnetic field). To minimize the systematic error associated with the Coulomb interaction between the two ions, they are kept about 1 millimeter apart from each other, resulting in fractional uncertainty below 1 × 10
–11
. Such precision opens the door to numerous applications of mass spectrometry, including metrology, fundamental physics, and weighing chemical bonds.
Defining the Epithelial Stem Cell Niche in Skin
Authors: Tumbar Tudorita , Guasch Geraldine , Greco Valentina , Blanpain Cedric , Lowry William E. , Rendl Michael , Fuchs Elaine
Published: December 2003

Many adult regenerative cells divide infrequently but have high proliferative capacity. We developed a strategy to fluorescently label slow-cycling cells in a cell type–specific fashion. We used this method to purify the label-retaining cells (LRCs) that mark the skin stem cell (SC) niche. We found that these cells rarely divide within their niche but change properties abruptly when stimulated to exit. We determined their transcriptional profile, which, when compared to progeny and other SCs, defines the niche. Many of the >100 messenger RNAs preferentially expressed in the niche encode surface receptors and secreted proteins, enabling LRCs to signal and respond to their environment.
Epigenetic Dynamics of Imprinted X Inactivation During Early Mouse Development
Authors: Okamoto Ikuhiro , Otte Arie P. , Allis C. David , Reinberg Danny , Heard Edith
Published: December 2003

The initiation of X-chromosome inactivation is thought to be tightly correlated with early differentiation events during mouse development. Here, we show that although initially active, the paternal X chromosome undergoes imprinted inactivation from the cleavage stages, well before cellular differentiation. A reversal of the inactive state, with a loss of epigenetic marks such as histone modifications and polycomb proteins, subsequently occurs in cells of the inner cell mass (ICM), which give rise to the embryo-proper in which random X inactivation is known to occur. This reveals the remarkable plasticity of the X-inactivation process during preimplantation development and underlines the importance of the ICM in global reprogramming of epigenetic marks in the early embryo.
The Preferred Substrate for RecA-Mediated Cleavage of Bacteriophage 434 Repressor Is the DNA-Bound Dimer
Authors: Pawlowski David R. , Koudelka Gerald B.
Published: December 2003

Induction of a lysogen of a lambdoid bacteriophage usually involves RecA-stimulated autoproteolysis of the bacteriophage repressor protein. Previous work on the phage repressors showed that the monomeric form of the protein is the target of RecA. Our previous work indicated that in the case of bacteriophage 434, virtually none of the repressor is present as a monomer in vivo. Hence, if the repressor in a lysogen is present as a dimer, how can RecA-stimulated autoproteolysis play a role in bacteriophage induction? We examined this question by determining the rate of RecA-stimulated 434 repressor cleavage as a function of repressor concentration and added DNA. Our results show that binding of 434 repressor to a specific DNA binding site dramatically increases the velocity of repressor autocleavage compared to the velocity of cleavage of the monomer and concentration-induced dimer. DNA binding-deficient hemidimers formed between the intact repressor and its C-terminal domain fragment have a lower rate of cleavage than DNA-bound dimers. These results show that the DNA-bound 434 repressor dimer, which is the form of the repressor that is required for its transcriptional regulatory functions, is the preferred form for RecA-stimulated autocleavage. We also show that the rate of repressor autocleavage is influenced by the sequence of the bound DNA. Kinetic analysis of the autocleavage reaction indicated that the DNA sequence influences the velocity of 434 repressor autocleavage by affecting the affinity of the repressor-DNA complex for RecA, not the chemical cleavage step. Regardless of the mechanism, the finding that the presence and precise sequence of DNA modulate the autocleavage reaction shows that DNA allosterically affects the function of 434 repressor.
Genomic Interrogation of the Dassie Bacillus Reveals It as a Unique RD1 Mutant within the
Mycobacterium tuberculosis
Complex
Authors: Mostowy Serge , Cousins Debby , Behr Marcel A.
Published: December 2003

Despite their remarkable genetic homology, members of the
Mycobacterium tuberculosis
complex express very different phenotypes, most notably in their spectra of clinical presentation. For example,
M. tuberculosis
is regarded as pathogenic to humans, whereas members having deleted RD1, such as
Mycobacterium microti
and
Mycobacterium bovis
BCG, are not. The dassie bacillus, an infrequent variant of the
M. tuberculosis
complex characterized as being most similar to
M. microti
, is the causative agent of tuberculosis (TB) in the dassie (
Procavia capensis
). Intriguingly, the dassie bacillus is not pathogenic to rabbits or guinea pigs and has never been documented to infect humans. Although it was identified more than a half-century ago, the reasons behind its attenuation are unknown. Because large sequence polymorphisms have presented themselves as the most obvious genomic distinction among members of the
M. tuberculosis
complex, the DNA content of the dassie bacillus was interrogated by Affymetrix GeneChip to identify regions that are absent from it but present in
M. tuberculosis
H37Rv. Comparison has led to the identification of nine regions of difference (RD), five of which are shared with
M. microti
(RDs 3, 7, 8, 9, and 10). Although the dassie bacillus does not share the other documented deletions in
M. microti
(RD1
mic
, RD5
mic
, MID1, MID2, and MID3), it has endured unique deletions in the regions of RD1, RD5, N-RD25, and Rv3081-Rv3082c (
virS
). RD1
das
, affecting only Rv3874-Rv3877, is the smallest natural deletion of the RD1 region uncovered and points to genes within this region that are likely implicated in virulence. Newfound deletions from the dassie bacillus are discussed in relation to their evolutionary and biological significance.
Natural Selection and Evolution of Streptococcal Virulence Genes Involved in Tissue-Specific Adaptations
Authors: Kalia Awdhesh , Bessen Debra E.
Published: December 2003

The molecular mechanisms underlying niche adaptation in bacteria are not fully understood. Primary infection by the pathogen group A streptococcus (GAS) takes place at either the throat or the skin of its human host, and GAS strains differ in tissue site preference. Many skin-tropic strains bind host plasminogen via the plasminogen-binding group A streptococcal M protein (PAM) present on the cell surface; inactivation of genes encoding either PAM or streptokinase (a plasminogen activator) leads to loss of virulence at the skin. Unlike PAM, which is present in only a subset of GAS strains, the gene encoding streptokinase (ska) is present in all GAS isolates. In this study, the evolution of the virulence genes known to be involved in skin infection was examined. Most genetic diversity withinskagenes was localized to a region encoding the plasminogen-docking domain (β-domain). The gene encoding PAM displayed strong linkage disequilibrium (P≪ 0.01) with a distinct phylogenetic cluster of theskaβ-domain-encoding region. Yet,skaalleles of distant taxa showed a history of intragenic recombination, and high intrinsic levels of recombination were found among GAS strains having different tissue tropisms. The data suggest that tissue-specific adaptations arise from epistatic coselection of bacterial virulence genes. Additional analysis ofskagenes showed that ∼4% of the codons underwent strong diversifying selection. Horizontal acquisition of oneskalineage from a commensalStreptococcusdonor species was also evident. Together, the data suggest that new phenotypes can be acquired through interspecies recombination between orthologous genes, while constrained functions can be preserved; in this way, orthologous genes may provide a rich and ready source for new phenotypes and thereby play a facilitating role in the emergence of new niche adaptations in bacteria.
Ruminococcus albus
8 Mutants Defective in Cellulose Degradation Are Deficient in Two Processive Endocellulases, Cel48A and Cel9B, Both of Which Possess a Novel Modular Architecture
Authors: Devillard Estelle , Goodheart Dara B. , Karnati Sanjay K. R. , Bayer Edward A. , Lamed Raphael , Miron Joshua , Nelson Karen E. , Morrison Mark
Published: December 2003

The cellulolytic bacterium
Ruminococcus albus
8 adheres tightly to cellulose, but the molecular biology underpinning this process is not well characterized. Subtractive enrichment procedures were used to isolate mutants of
R. albus
8 that are defective in adhesion to cellulose. Adhesion of the mutant strains was reduced 50% compared to that observed with the wild-type strain, and cellulose solubilization was also shown to be slower in these mutant strains, suggesting that bacterial adhesion and cellulose solubilization are inextricably linked. Two-dimensional polyacrylamide gel electrophoresis showed that all three mutants studied were impaired in the production of two high-molecular-mass, cell-bound polypeptides when they were cultured with either cellobiose or cellulose. The identities of these proteins were determined by a combination of mass spectrometry methods and genome sequence data for
R. albus
8. One of the polypeptides is a family 9 glycoside hydrolase (Cel9B), and the other is a family 48 glycoside hydrolase (Cel48A). Both Cel9B and Cel48A possess a modular architecture, Cel9B possesses features characteristic of the B
2
(or theme D) group of family 9 glycoside hydrolases, and Cel48A is structurally similar to the processive endocellulases CelF and CelS from
Clostridium cellulolyticum
and
Clostridium thermocellum
, respectively. Both Cel9B and Cel48A could be recovered by cellulose affinity procedures, but neither Cel9B nor Cel48A contains a dockerin, suggesting that these polypeptides are retained on the bacterial cell surface, and recovery by cellulose affinity procedures did not involve a clostridium-like cellulosome complex. Instead, both proteins possess a single copy of a novel X module with an unknown function at the C terminus. Such X modules are also present in several other
R. albus
glycoside hydrolases and are phylogentically distinct from the fibronectin III-like and X modules identified so far in other cellulolytic bacteria.
In
Pseudomonas syringae
pv. phaseolicola, Expression of the
argK
Gene, Encoding the Phaseolotoxin-Resistant Ornithine Carbamoyltransferase, Is Regulated Indirectly by Temperature and Directly by a Precursor Resembling Carbamoylphosphate
Authors: López-López Karina , Hernández-Flores José Luis , Cruz-Aguilar Marisa , Alvarez-Morales Ariel
Published: December 2003

Pseudomonas syringae
pv. phaseolicola synthesizes a non-host-specific toxin, phaseolotoxin, and also synthesizes a phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) to protect itself from its own toxin. ROCT is encoded by
argK
, which is expressed coordinately with phaseolotoxin synthesis at 18°C. To investigate the regulatory mechanisms of this system, null mutants were constructed for
argK
,
argF
(encoding the phaseolotoxin-sensitive OCTase [SOCT]), and
amtA
(encoding an amidinotransferase involved in phaseolotoxin synthesis). The
argF
mutant did not exhibit arginine auxotrophy when grown in M9 medium at 28°C, because under this condition SOCT was replaced by ROCT. This loss of thermoregulation of
argK
was apparently caused by accumulation of carbamoylphosphate, one of the substrates of SOCT. Carbamoylphosphate, which has a structure similar to that of the inorganic moiety of phaseolotoxin, was used in induction assays with wild-type
P. syringae
pv. phaseolicola and was shown to be able to induce
argK
expression in M9 medium at 28°C. These results indicate that
argK
expression is independent of temperature and is regulated directly by a compound resembling the inorganic moiety of phaseolotoxin.
Esp-Independent Biofilm Formation by
Enterococcus faecalis
Authors: Kristich Christopher J. , Li Yung-Hua , Cvitkovitch Dennis G. , Dunny Gary M.
Published: December 2003

Enterococcus faecalis
is a gram-positive opportunistic pathogen known to form biofilms in vitro. In addition, this organism is often isolated from biofilms on the surfaces of various indwelling medical devices. However, the molecular mechanisms regulating biofilm formation in these clinical isolates are largely unknown. Recent work has suggested that a specific cell surface protein (Esp) of
E. faecalis
is critical for biofilm formation by this organism. However, in the same study,
esp
-deficient strains of
E. faecalis
were found to be capable of biofilm formation. To test the hypothesis that Esp is dispensable for biofilm formation by
E. faecalis
, we used microtiter plate assays and a chemostat-based biofilm fermentor assay to examine biofilm formation by genetically well-defined, non-Esp-expressing strains. Our results demonstrate that in vitro biofilm formation occurs, not only in the absence of
esp
, but also in the absence of the entire pathogenicity island that harbors the
esp
coding sequence. Using scanning electron microscopy to evaluate biofilms of
E. faecalis
OG1RF grown in the fermentor system, biofilm development was observed to progress through multiple stages, including attachment of individual cells to the substratum, microcolony formation, and maturation into complex multilayered structures apparently containing water channels. Microtiter plate biofilm analyses indicated that biofilm formation or maintenance was modulated by environmental conditions. Furthermore, our results demonstrate that expression of a secreted metalloprotease, GelE, enhances biofilm formation by
E. faecalis
. In summary,
E. faecalis
forms complex biofilms by a process that is sensitive to environmental conditions and does not require the Esp surface protein.
Formation and Composition of the
Bacillus anthracis
Endospore
Authors: Liu Hongbin , Bergman Nicholas H. , Thomason Brendan , Shallom Shamira , Hazen Alyson , Crossno Joseph , Rasko David A. , Ravel Jacques , Read Timothy D. , Peterson Scott N. , Yates John , Hanna Philip C.
Published: December 2003

The endospores of
Bacillus anthracis
are the infectious particles of anthrax. Spores are dormant bacterial morphotypes able to withstand harsh environments for decades, which contributes to their ability to be formulated and dispersed as a biological weapon. We monitored gene expression in
B. anthracis
during growth and sporulation using full genome DNA microarrays and matched the results against a comprehensive analysis of the mature anthrax spore proteome. A large portion (∼36%) of the
B. anthracis
genome is regulated in a growth phase-dependent manner, and this regulation is marked by five distinct waves of gene expression as cells proceed from exponential growth through sporulation. The identities of more than 750 proteins present in the spore were determined by multidimensional chromatography and tandem mass spectrometry. Comparison of data sets revealed that while the genes responsible for assembly and maturation of the spore are tightly regulated in discrete stages, many of the components ultimately found in the spore are expressed throughout and even before sporulation, suggesting that gene expression during sporulation may be mainly related to the physical construction of the spore, rather than synthesis of eventual spore content. The spore also contains an assortment of specialized, but not obviously related, metabolic and protective proteins. These findings contribute to our understanding of spore formation and function and will be useful in the detection, prevention, and early treatment of anthrax. This study also highlights the complementary nature of genomic and proteomic analyses and the benefits of combining these approaches in a single study.
Fine-Tuning in Regulation of Clp Protein Content inBacillus subtilis
Authors: Gerth Ulf , Kirstein Janine , Mostertz Jörg , Waldminghaus Torsten , Miethke Marcus , Kock Holger , Hecker Michael
Published: December 2003

Clp-controlled proteolysis inBacillus subtilisseems to play a substantial role, particularly under stress conditions. Calibrated Western blot analyses were used to estimate the approximate numbers of heat-inducible Clp molecules within a single cell. According to these numbers, the different Clp ATPases do not seem to compete for the proteolytic subunit ClpP. Coimmunoprecipitation experiments revealed the predicted specific ClpX-ClpP, ClpC-ClpP, and ClpE-ClpP interactions. ClpE and ClpX are rapidly degraded in wild-type cells during permanent heat stress but remained almost stable in aclpPmutant, suggesting ClpP-dependent degradation. In particular, ClpCP appeared to be involved in the degradation of the short-lived ClpE ATPase, indicating a negative “autoregulatory” circuit for this particular Clp ATPase at the posttranslational level. Analysis of the half-life of stress-inducibleclpmRNAs during exponential growth and heat shock revealed precise regulation of the synthesis of each Clp protein at the posttranscriptional level as well to meet the needs ofB. subtilis.
Surfaces of Spo0A and RNA Polymerase Sigma Factor A That Interact at the
spoIIG
Promoter in
Bacillus subtilis
Authors: Kumar Amrita , Buckner Starke Cindy , DeZalia Mark , Moran Charles P.
Published: December 2003

In
Bacillus subtilis
, the DNA binding protein Spo0A activates transcription from two classes of promoters, those used by RNA polymerase containing the primary sigma factor, σ
A
(e.g.,
spoIIG
), and those used by RNA polymerase containing the secondary sigma factor, σ
H
(e.g.,
spoIIA
). Several single amino acid substitutions in region 4 of σ
A
define positions in σ
A
that are specifically required for Spo0A-dependent promoter activation. Similarly, several single amino acid substitutions in Spo0A define positions in Spo0A that are required for σ
A
-dependent promoter activation but not for other functions of Spo0A. It is unknown whether these amino acids in Spo0A interact directly with those in region 4 of σ
A
or whether they interact with another subunit of RNA polymerase to effect promoter activation. Here we report the identification of a new amino acid in region 4 of σ
A
, arginine at position 355 (R355), that is involved in Spo0A-dependent promoter activation. To further investigate the role of R355, we used the coordinates of Spo0A and sigma region 4, each in complex with DNA, to build a model for the interaction of σ
A
and Spo0A at the
spoIIG
promoter. We tested the model by examining the effects of amino acid substitutions in the putative interacting surfaces of these molecules. As predicted by the model, we found genetic evidence for interaction of R355 of σ
A
with glutamine at position 221 of Spo0A. These results appear to define the surfaces of Spo0A and σ
A
that directly interact during activation of the
spoIIG
promoter.
Effect of the CopB Auxiliary Replication Control System on Stability of Maintenance of Par
+
Plasmid R1
Authors: Olsson Jan A. , Paulsson Johan , Nordström Kurt
Published: December 2003

Plasmid R1 is a low-copy-number plasmid that is present at a level of about four or five copies per average cell. The copy number is controlled posttranscriptionally at the level of synthesis of the rate-limiting initiator protein RepA. In addition to this, R1 has an auxiliary system that derepresses a second promoter at low copy numbers, leading to increased
repA
mRNA synthesis. This promoter is normally switched off by a constitutively synthesized plasmid-encoded repressor protein, CopB; in cells with low copy numbers, the concentration of CopB is low and the promoter is derepressed. Here we show that the rate of loss of a Par
+
derivative of the basic replicon of R1 increased about sevenfold when the cells contained a high concentration of the CopB protein formed from a compatible plasmid.