bluuebunny/crossref_metadata_2025 huggingface dataset:
EDL and soleus muscles of the C57BL6J/dy2j laminin‐α2‐deficient dystrophic mouse are not vulnerable to eccentric contractions
Authors: Head Stewart I. , Bakker Anthony J. , Liangas Georgios
Published: June 2004

Many muscular dystrophies arise as a consequence of mutations in a series of interconnected proteins associated with the sarcolemma. This group of proteins is collectively referred to as the ‘dystrophin‐associated complex’. We used the C57BL6J/dy2j, dystrophia muscularis, dystrophic mouse, in which the laminin‐α2 component of the dystrophin‐associated complex is mutated, to test the hypothesis that the disruption of this complex will destabilize the lipid bilayer, rendering it more susceptible to damage during eccentric contractions. We demonstrated that neither slow‐ nor fast‐twitch dystrophic muscles were more susceptible to eccentric contractions when compared with controls. Only fast‐twitch extensor digitorum longus (EDL) muscles (from both dystrophic and control mice) showed an irreversible loss of force with our eccentric contraction protocol, suggesting that it is the fast 11b fibres (not present in slow‐twitch soleus) which are most susceptible to eccentric damage. We used the general anaesthetic halothane to increase the fluidity of the lipid bilayer to see if this would uncover any greater susceptibility of the dystrophic muscle to eccentric damage. This also did not reveal any greater fragility of fast‐ and slow‐twitch dystrophic muscles. We did, however, demonstrate that halothane made both control and dystrophic fast‐ and slow‐twitch muscles more susceptible to eccentric contraction damage. The C57BL6J/dy2j dystrophic laminopathy produced the pathophysiological and pathohistological characteristics associated with muscular dystrophy: the fast‐ and slow‐twitch dystophic muscles produced only 55 and 53%, respectively, of the force of control muscles and 34 and 40%, respectively, of the dystrophic muscle fibres were branched. The presence of the branched fibres in the dystrophic muscles did not make them more susceptible to eccentric damage but may have contributed to the reduction in maximal force in the dystrophic muscles. We conclude that our data do not support the structural hypothesis that the dystrophin‐associated complex acts as a scaffolding to support the lipid bilayer, but are consistent with channel‐based hypotheses put forward to explain the dystrophic process.
Analysis of ex vivo left ventricular pressure–volume relations in the isolated murine ejecting heart
Authors: Grieve David J. , Cave Alison C. , Byrne Jonathan A. , Layland Joanne , Shah Ajay M.
Published: June 2004

The development of microconductance technology to study cardiac pressure–volume relations in mice in vivo has significantly advanced the haemodynamic assessment of gene‐modified models of cardiovascular disease. In this study, we describe the application of microconductance analysis of cardiac function to the isolated murine ejecting heart. This ex vivo model is complementary to the previously described in vivo preparation, allows assessment without confounding effects of anaesthetic or neurohumoral influences and enables careful control of cardiac loading (particularly preload). Ex vivo pressure–volume relations in the isolated murine heart are sensitive to changes in myocardial contractility induced by β‐adrenoceptor stimulation or β‐adrenoceptor blockade, as well as the effects of chronic pressure overload induced by aortic banding. We present data for both steady‐state analyses of the Frank–Starling relation and for assessment of the left ventricular pressure–volume relation over variably loaded beats, which allows investigation of the end‐systolic and end‐diastolic pressure–volume relations. The measurement of ventricular volume in addition to pressure under carefully controlled loading conditions in the isolated ejecting heart allows a comprehensive analysis of cardiac contractile function, and provides a useful complementary model for the assessment of cardiac performance in murine models of heart disease.
Orthostatic tolerance and blood volumes in Andean high altitude dwellers
Authors: E. Claydon V. , J. Norcliffe L. , P. Moore J. , Rivera‐Ch M. , Leon‐Velarde F. , Appenzeller O. , Hainsworth R.
Published: June 2004

Orthostatic tolerance is a measure of the ability to prevent hypotension during gravitational stress. It is known to be dependent on the degree of vasoconstriction and the magnitude of plasma volume, but the possible influence of packed cell volume (PCV) is unknown. High altitude residents have high haematocrits and probably high packed cell volumes. However, it is not known whether plasma volume and blood volume are affected, or whether their orthostatic tolerance is different from low altitude residents. In this study we determined plasma volume, PCV and orthostatic tolerance in a group of high altitude dwellers (HA), including a subgroup of highland dwellers with chronic mountain sickness (CMS) and extreme polycythaemia. Plasma volume and PCV were determined using Evans Blue dye dilution and peripheral haematocrit. Orthostatic tolerance was assessed as the time to presyncope in a test of head‐up tilting and lower body suction. All studies were performed at 4338 m. Results showed that plasma volumes were not significantly different between CMS and HA, or in highland dwellers compared to those seen previously in lowlanders. PCV and haematocrit were greater in CMS than in HA. Orthostatic tolerance was high in both CMS and HA, although the heart rate responses to orthostasis were smaller in CMS than HA. Orthostatic tolerance was correlated with haematocrit (r= 0.57, P < 0.01) and PCV (r= 0.54, P < 0.01). This investigation has shown that although high altitude residents have large PCV, their plasma volumes were similar to lowland dwellers. The group with CMS have a particularly large PCV and also have a very high orthostatic tolerance, despite smaller heart rate responses. These results are compatible with the view that PCV is of importance in determining orthostatic tolerance.
Gut hormones in the control of appetite
Authors: Murphy K.G. , Bloom S.R.
Published: June 2004

Obesity is one of the greatest threats to the health of the developed world. In order to design effective drugs to treat the alarming increase in obesity, it is essential to understand the physiology of normal appetite control and the pathophysiology of obesity. The hypothalamus interprets and integrates neural and humoral inputs to provide a coordinated feeding and energy expenditure response. Recent evidence suggests that certain gut hormones – ghrelin, polypeptide YY, pancreatic polypeptide, glucagon‐like‐peptide 1 and oxyntomodulin – have a physiological role in governing satiety via the hypothalamus. Gut hormone appetite‐regulatory systems represent a potential target for the design of antiobesity drugs.
Light‐evoked current responses in rod bipolar cells, cone depolarizing bipolar cells and AII amacrine cells in dark‐adapted mouse retina
Authors: Pang Ji‐Jie , Gao Fan , Wu Samuel M.
Published: June 2004

Light‐evoked excitatory cation current (ΔIC) and inhibitory chloride current (ΔICl) of rod and cone depolarizing bipolar cells (DBCRs and DBCCs) and AII amacrine cells (AIIACs) in dark‐adapted mouse retinal slices were studied by whole‐cell voltage‐clamp recording techniques, and the cell morphology was revealed by Lucifer yellow fluorescence with a confocal microscope. ΔIC of all DBCRs exhibited similar high sensitivity to 500 nm light, but two patterns of ΔICl were observed in DBCRs with slightly different axon morphology. At least two types of DBCCs were identified: one with axon terminals ramified in 70–85% of the depth of the inner plexiform layer (IPL) and DBCR‐like ΔIC sensitivity, whereas the other with axon terminals ramified in 55–75% of IPL depth and much lower ΔIC sensitivity. The relative rod/cone inputs to DBCs and AIIACs were analysed by comparing the ΔIC and ΔICl thresholds and dynamic ranges with the corresponding values of rods and cones. On average, the sensitivity of a DBCR to the 500 nm light is about 20 times higher than that of a rod. The sensitivity of an AIIAC is more than 1000 times higher than that of a rod, suggesting that AIIAC responses are pooled through a coupled network of about 40 AIIACs. Interactions of rod and cone signals in dark‐adapted mouse retina appear asymmetrical: rod signals spread into the cone system more efficiently than cone signals into the rod system. The mouse synaptic circuitry allows small rod signals to be highly amplified, and effectively transmitted to the cone system via rod–cone and AIIAC–DBCC coupling.
Probing the pore of the auditory hair cell mechanotransducer channel in turtle
Authors: Farris H. E. , LeBlanc C. L. , Goswami J. , Ricci A. J.
Published: June 2004

Hair cell mechano‐electric transducer (MET) channels play a pivotal role in auditory and vestibular signal detection, yet few data exist regarding their molecular nature. Present work characterizes the MET channel pore, a region whose properties are thought to be intrinsically determined. Two approaches were used. First, the channel was probed with antagonists of candidate channel subtypes including: cyclic nucleotide‐gated channels, transient receptor potential channels and gap‐junctional channels. Eight new antagonists were identified. Most of the effective antagonists had a partially charged amine group predicted to penetrate the channel pore, antagonizing current flow, while the remainder of the molecule prevented further permeation of the compound through the pore. This blocking mechanism was tested using curare to demonstrate the open channel nature of the block and by identifying methylene blue as a permeant channel blocker. The second approach estimated dimensions of the channel pore with simple amine compounds. The narrowest diameter of the pore was calculated as 12.5 ± 0.8  Å and the location of a binding site ∼45% of the way through the membrane electric field was calculated. Channel length was estimated as ∼31 Å and the width of the pore mouth at < 17  Å. Each effective antagonist had a minimal diameter, measured about the penetrating amine, of less than the pore diameter, with a direct correlation between IC50 and minimal diameter. The IC50 was also directly related to the length of the amine side chains, further validating the proposed pore blocking mechanism. Data provided by these two approaches support a hypothesis regarding channel permeation and block that incorporates molecular dimensions and ion interactions within the pore.
Characterization of release‐independent short‐term depression in the juvenile rat hippocampus
Authors: Muñoz‐Cuevas J. , Vara H. , Colino A.
Published: June 2004

Short‐term depression strongly influences neuronal activity in cerebral circuits and contributes to low‐pass temporal filtering of information. In this work, we show that synaptic depression evoked by stimulation of commissural–Schaffer collateral afferents at 10 Hz is associated with a reduction of the fibre volley. This depression of action potentials is also evident in the absence of extracellular Ca2+, which underlies its release‐independent nature. In addition, this reduction of the excitability is independent of failures in action potential propagation since increasing the distance between the stimulus and recording electrodes does not alter this effect. Whole‐cell recordings show that tetanic stimulation at supraminimal intensity induces action potential failures preceded by changes in the repolarization rate of the action potentials leading the membrane potential to hyperpolarized values. This activity‐dependent hyperpolarization was blocked by ouabain, an indication of the important role of the Na+–K+‐ATPase in this process. Then again, an alteration of the firing threshold was observed when action potentials were elicited either by somatic current injection or by synaptic stimulation, which indicates that this mechanism could alter the EPSP–spike coupling in these cells. The results suggest that these factors act together to reduce gradually the safety factor for action potential generation and to produce failures in action potential initiation; in fact, experiments made at twice the supraminimal intensity show a dramatic decrease in the rate of these failures. Taken together, the results suggest the existence of a release‐independent component of short‐term depression that is related to failures in action potential initiation.
Molecular basis of slow activation of the human ether‐á‐go‐go related gene potassium channel
Authors: Subbiah Rajesh N. , Clarke Catherine E. , Smith David J. , Zhao JingTing , Campbell Terence J. , Vandenberg Jamie I.
Published: June 2004

The human ether‐á‐go‐go related gene (HERG) encodes the pore forming α‐subunit of the rapid delayed rectifier K+ channel which is central to the repolarization phase of the cardiac action potential. HERG K+ channels have unusual kinetics characterized by slow activation and deactivation, yet rapid inactivation. The fourth transmembrane domain (S4) of HERG, like other voltage‐gated K+ channels, contains multiple positive charges and is the voltage sensor for activation. In this study, we mutated each of the positively charged residues in this region to glutamine (Q), expressed the mutant and wild‐type (WT) channels in Xenopus laevis oocytes and studied them using two‐electrode voltage clamp methods. K525Q channels activated at more hyperpolarized potentials than WT, whereas all the other mutant channels activated at more depolarized potentials. All mutants except for R531Q also had a reduction in apparent gating charge associated with activation. Mutation of K525 to cysteine (C) resulted in a less dramatic phenotype than K525Q. The addition of the positively charged MTSET to K525C altered the phenotype to one more similar to K525Q than to WT. Therefore it is not charge per se, but the specific lysine side chain at position 525, that is crucial for stabilizing the closed state. When rates of activation and deactivation for WT and mutant channels were compared at equivalent total (chemical + electrostatic) driving forces, K525Q and R528Q accelerated activation but had no effect on deactivation, R531Q slowed activation and deactivation, R534Q accelerated activation but slowed deactivation and R537Q accelerated deactivation but had no effect on activation. The main conclusions we can draw from these data are that in WT channels K525 stabilizes the closed state, R531 stabilizes the open state and R534 participates in interactions that stabilize pre‐open closed states.
Human forearm position sense after fatigue of elbow flexor muscles
Authors: Walsh L. D. , Hesse C. W. , Morgan D. L. , Proske U.
Published: June 2004

After a period of eccentric exercise of elbow flexor muscles of one arm in young, adult human subjects, muscles became fatigued and damaged. Damage indicators were a fall in force, change in resting elbow angle and delayed onset of soreness. After the exercise, subjects were asked to match the forearm angle of one arm, whose position was set by the experimenter, with their other arm. Subjects matched the position of the unsupported reference arm, when this was unexercised, with a significantly more flexed position in their exercised indicator arm. Errors were in the opposite direction when the reference arm was exercised. The size of the errors correlated with the drop in force. Less consistent errors were observed when the reference arm was supported. A similar pattern of errors was seen after concentric exercise, which does not produce muscle damage. The data suggested that subjects were using as a position cue the perceived effort required to maintain a given forearm angle against the force of gravity. The fall in force from fatigue after exercise meant more effort was required to maintain a given position. That led to matching errors between the exercised and unexercised arms. It was concluded that while a role for muscle spindles in kinaesthesia cannot be excluded, detailed information about static limb position can be derived from the effort required to support the limb against the force of gravity.
Expression of several cytoskeletal proteins in ovine cerebral arteries: developmental and functional considerations
Authors: Zhao Yu , Xiao Harvey , Long Wen , Pearce William J. , Longo Lawrence D.
Published: June 2004

Cytoskeleton proteins play important roles in regulating vascular smooth muscle (VSM) contraction and relaxation. We tested the hypotheses that the expression levels of several of these proteins change significantly during the course of development, and that these changes contribute to age‐related changes in contractile responses. In cerebral arteries from 95‐day (d) gestation and 140‐d fetus, newborn lambs, and adult sheep, by Western immunoblot (n= 5 for each age) we quantified the relative expression of α‐actin, α‐tubulin, cyclophilin A, and proliferating cell nuclear antigen (PCNA). In addition, we examined middle cerebral artery tension responses to phenylephrine (PHE) stimulation in the absence or presence of cytochalasin D (3 × 10−7m) and nocodazole (3 × 10−6m), inhibitors of α‐actin and α‐tubulin polymerization, respectively. The expression levels of α‐actin and cyclophilin A varied little during the course of development. In contrast, α‐tubulin expression was ∼2.5‐fold greater in both fetal age groups as compared to adult. Also, as compared to adult and as expected, expression of PCNA was several‐fold greater in cerebral arteries of the 95‐d fetus (×8), 140‐d fetus (×5), and newborn (×3). In both adult and fetal middle cerebral artery, cytochalasin D‐induced inhibition of actin polymerization decreased PHE‐induced contraction, to ∼60 and ∼40% of control, respectively (despite no significant change in expression level). In contrast, α‐tubulin inhibition by nocodazole showed little effect on PHE‐induced tension (in spite of the age‐related decrease in expression). In conclusion, expression levels of α‐actin, a thin filament protein involved in contraction, remained relatively constant during the course of development, as did the effects of inhibition of its polymerization on contractility. In contrast, α‐tubulin, important in intracellular protein trafficking, showed a significant age‐related decrease in expression and played a relatively minor role in contractility. The present studies suggest that other cytoskeletal structural proteins and/or elements of pharmaco‐mechanical coupling are important to developmental differences in cerebrovascular contractility. In addition, the relatively constant expression levels of α‐actin and cyclophilin A with development, suggest that these are useful internal standards for studies of cytosolic protein expression.
Expression of the sodium–myo‐inositol cotransporter SMIT2 at the apical membrane of Madin‐Darby canine kidney cells
Authors: Bissonnette Pierre , Coady Michael J. , Lapointe Jean‐Yves
Published: June 2004

Myo‐inositol is a compatible osmolyte used by cells which are challenged by variations in extracellular osmolarity, as in the renal medulla. In order to accumulate large quantities of this polyol, cells rely on Na+‐dependent transporters such as SMIT1. We have recently identified a second Na+–myo‐inositol cotransporter, SMIT2, which presents transport characteristics corresponding to those recently described for the apical membrane of renal proximal tubules. In order to further characterize this transport system, we transfected Madin‐Darby canine kidney (MDCK) cells with rabbit SMIT2 cDNA and selected a stable clone with a high expression level. The accumulation of radiolabelled myo‐inositol by this cell line is 20‐fold larger than that seen in native MDCK cells. The affinity for myo‐inositol of MDCK cells transfected with SMIT2 is slightly lower (Km= 334 μm) than that found in voltage‐clamped Xenopus laevis oocytes expressing SMIT2 (Km= 120 μm). Transport studies performed using semipermeable filters showed complete apical targeting of the SMIT2 transporter. This apical localization of SMIT2 was confirmed by transport studies on purified rabbit renal brush border membrane vesicles (BBMVs). Using a purified antibody against SMIT2, we were also able to detect the SMIT2 protein (molecular mass = 66 kDa) in Western blots of BBMVs purified from SMIT2‐transfected MDCK cells. SMIT2 activity was also shown to be stimulated 5‐fold when submitted to 24 h hypertonic treatment (+200 mosmol l−1). The SMIT2‐MDCK cell line thus appears to be a promising model for studying SMIT2 biochemistry and regulation.
Relaxation from a voluntary contraction is preceded by increased excitability of motor cortical inhibitory circuits
Authors: Buccolieri Alessandro , Abbruzzese Giovanni , Rothwell John C.
Published: June 2004

Termination of a muscle contraction is as important a part of movement as muscle activation yet the mechanisms responsible are less well understood. In the present experiments we examined the possible role of intracortical inhibitory circuits in terminating a 20% maximum isometric contraction of the first dorsal interosseous muscle (FDI) in eight healthy subjects. Subjects performed the task simultaneously with both hands and received single or pairs (at an interstimulus interval of 3 ms to evaluate short interval intracortical inhibition, SICI) of transcranial magnetic stimuli (TMS) via a focal coil over the motor hand area of the left hemisphere at different times before and after the onset of relaxation. The amplitude of the motor‐evoked potential (MEP) following a single or a pair of TMS pulses was measured in the right FDI and plotted relative to the onset of relaxation as estimated from the surface electromyogram (EMG) of the left FDI. MEPs were larger during contraction than after relaxation whereas SICI was absent during contraction and reappeared after relaxation. We found that in all subjects, the time course of MEP changes during relaxation was closely fitted by a Boltzmann sigmoidal curve which allowed us to estimate the mean MEP amplitudes as well as the ratio of the amplitudes after single or pairs of TMS pulses (i.e.%SICI) at any time in the task. The data showed that the amplitude of MEPs to single pulse TMS had started to decline at about the same time as the onset of EMG silence. Furthermore, the size of the MEPs evoked by paired pulses decreased up to 30 ms beforehand. The latter suggests that an increase in SICI occurs prior to the onset of MEP changes, and hence that increased cortical inhibition may play a role in suppressing corticospinal excitability during relaxation. A subsidiary experiment showed that the time relations of changes in SICI and MEP were unchanged by a period of 10 min training on the task.
Altered neurotransmitter control of reflex vasoconstriction in aged human skin
Authors: Thompson Caitlin S. , Kenney W. Larry
Published: June 2004

Cutaneous vasoconstriction (VC) in response to cooling is attenuated in older humans; however, mechanisms underlying this functional decline remain unclear. The present study tested the hypothesis that the contributions of noradrenaline (NA) and sympathetic cotransmitters to reflex‐mediated cutaneous VC are altered with age. In 11 young (18–26 years) and 11 older (61–77 years) men and women, forearm skin blood flow was monitored at three sites using laser Doppler flowmetry (LDF) while mean skin temperature was lowered from 34 to 30.5°C using a water‐perfused suit. Cutaneous vascular conductance (CVC; LDF/mean arterial pressure) was expressed as percentage change from baseline (%ΔCVCbase). Solutions of yohimbine + propranolol (Y + P), bretylium tosylate (BT), and lactated Ringer solution were infused via intradermal microdialysis at each LDF site to antagonize α‐ and β‐adrenoceptors, block sympathetic release of NA and cotransmitters, and act as control, respectively. During cooling, VC was attenuated at the control site in older subjects compared to young subjects (−16 ± 3 versus−34 ± 4%ΔCVCbase, P < 0.001). Y + P attenuated VC in young subjects (−13 ± 8%ΔCVCbase, P < 0.001 versus control) and abolished VC in older subjects (0 ± 3%ΔCVCbase, P > 0.9 versus baseline). BT completely blocked VC in both age groups. Cutaneous VC in young subjects is mediated by both NA and sympathetic cotransmitter(s); however, reflex VC in aged skin is attenuated compared to young and appears to be mediated solely by NA.
P2X receptor subtype‐specific modulation of excitatory and inhibitory synaptic inputs in the rat brainstem
Authors: Watano Tomokazu , Calvert Jennifer A. , Vial Catherine , Forsythe Ian D. , Evans Richard J.
Published: June 2004

The role of P2 receptors in synaptic transmission to the rat medial nucleus of the trapezoid body (MNTB) was studied in an in vitro brain slice preparation. Whole‐cell patch recordings were made and spontaneous synaptic responses studied under voltage clamp during application of P2X receptor agonists. ATPγS (100 μm) had no effect on holding current, but facilitated spontaneous excitatory postsynaptic current (sEPSC) frequency in 41% of recordings and facilitated spontaneous inhibitory postsynaptic currents (sIPSCs) in 20% of recordings. These were blocked by the P2 receptor antagonist suramin (100 μm). α,β‐meATP also facilitated sEPSC and sIPSC frequency, while l‐β,γ‐meATP facilitated only sIPSCs. The sEPSC facilitation by ATPγS was blocked by TTX (but did not block facilitation of sIPSCs). sEPSC facilitation was blocked by PPADS (30 μm) and the selective P2X3 receptor antagonist A‐317491 (3 μm), suggesting that modulation of sEPSCs involves P2X3 receptor subunits. α,β‐meATP‐facilitated sIPSCs were also recorded in wild‐type mouse MNTB neurones, but were absent in the MNTB from P2X1 receptor‐deficient mice demonstrating a functional role for P2X1 receptors in the CNS.
Postnatal Refinement of Peripheral Olfactory Projections
Authors: Zou Dong-Jing , Feinstein Paul , Rivers Aimée L. , Mathews Glennis A. , Kim Ann , Greer Charles A. , Mombaerts Peter , Firestein Stuart
Published: June 2004

Axonal projections from the olfactory epithelium to the olfactory bulb are organized into glomeruli according to the expressed odorant receptor. Using gene-targeted mice, we show that glomerular maturation proceeds along different time courses for two similar receptors and requires sensory input during distinct sensitive periods. During early development, some glomeruli are innervated by axons of neurons that do not express the same receptor. These heterogeneous glomeruli normally disappear with age, but they persist in adults deprived of sensory input by unilateral and permanent naris closure. Persistence may be due, in part, to prolonged survival of olfactory sensory neurons.
The Bacterial Condensin MukBEF Compacts DNA into a Repetitive, Stable Structure
Authors: Case Ryan B. , Chang Yun-Pei , Smith Steven B. , Gore Jeff , Cozzarelli Nicholas R. , Bustamante Carlos
Published: June 2004

Condensins are conserved proteins containing SMC (structural maintenance of chromosomes) moieties that organize and compact chromosomes in an unknown mechanism essential for faithful chromosome partitioning. We show that MukBEF, the condensin in
Escherichia coli
, cooperatively compacts a single DNA molecule into a filament with an ordered, repetitive structure in an adenosine triphosphate (ATP) binding–dependent manner. When stretched to a tension of ∼17 piconewtons, the filament extended in a series of repetitive transitions in a broad distribution centered on 45 nanometers. A filament so extended and held at a lower force recondensed in steps of 35 nanometers or its multiples; this cycle was repeatable even in the absence of ATP and free MukBEF. Remarkably, the pattern of transitions displayed by a given filament during the initial extension was identical in every subsequent extension. Hence, after being deformed micrometers in length, each filament returned to its original compact structure without the addition of energy. Incubation with topoisomerase I increased the rate of recondensation and allowed the structure to extend and reform almost reversibly, indicating that supercoiled DNA is trapped in the condensed structure. We suggest a new model for how MukBEF organizes the bacterial chromosome in vivo.
Small Bilaterian Fossils from 40 to 55 Million Years Before the Cambrian
Authors: Chen Jun-Yuan , Bottjer David J. , Oliveri Paola , Dornbos Stephen Q. , Gao Feng , Ruffins Seth , Chi Huimei , Li Chia-Wei , Davidson Eric H.
Published: June 2004

Ten phosphatized specimens of a small (<180 micrometers) animal displaying clear bilaterian features have been recovered from the Doushantuo Formation, China, dating from 40 to 55 million years before the Cambrian. Seen in sections, this animal (
Vernanimalcula guizhouena
gen. et sp. nov.) had paired coeloms extending the length of the gut; paired external pits that could be sense organs; bilateral, anterior-posterior organization; a ventrally directed anterior mouth with thick walled pharynx; and a triploblastic structure. The structural complexity is that of an adult rather than a larval form. These fossils provide the first evidence confirming the phylogenetic inference that Bilateria arose well before the Cambrian.
A New Biology for a New Century
Authors: Woese Carl R.
Published: June 2004

Biology today is at a crossroads. The molecular paradigm, which so successfully guided the discipline throughout most of the 20th century, is no longer a reliable guide. Its vision of biology now realized, the molecular paradigm has run its course. Biology, therefore, has a choice to make, between the comfortable path of continuing to follow molecular biology's lead or the more invigorating one of seeking a new and inspiring vision of the living world, one that addresses the major problems in biology that 20th century biology, molecular biology, could not handle and, so, avoided. The former course, though highly productive, is certain to turn biology into an engineering discipline. The latter holds the promise of making biology an even more fundamental science, one that, along with physics, probes and defines the nature of reality. This is a choice between a biology that solely does society's bidding and a biology that is society's teacher.
Compartmentalization of Gene Expression duringBacillus subtilisSpore Formation
Authors: Hilbert David W. , Piggot Patrick J.
Published: June 2004

Gene expression in members of the familyBacillaceaebecomes compartmentalized after the distinctive, asymmetrically located sporulation division. It involves complete compartmentalization of the activities of sporulation-specific sigma factors, σFin the prespore and then σEin the mother cell, and then later, following engulfment, σGin the prespore and then σKin the mother cell. The coupling of the activation of σFto septation and σGto engulfment is clear; the mechanisms are not. The σ factors provide the bare framework of compartment-specific gene expression. Within each σ regulon are several temporal classes of genes, and for key regulators, timing is critical. There are also complex intercompartmental regulatory signals. The determinants for σFregulation are assembled before septation, but activation follows septation. Reversal of the anti-σFactivity of SpoIIAB is critical. Only the origin-proximal 30% of a chromosome is present in the prespore when first formed; it takes ≈15 min for the rest to be transferred. This transient genetic asymmetry is important for prespore-specific σFactivation. Activation of σErequires σFactivity and occurs by cleavage of a prosequence. It must occur rapidly to prevent the formation of a second septum. σGis formed only in the prespore. SpoIIAB can blockσGactivity, but SpoIIAB control does not explain why σGis activated only after engulfment. There is mother cell-specific excision of an insertion element insigKand σE-directed transcription ofsigK, which encodes pro-σK. Activation requires removal of the prosequence following aσG-directed signal from the prespore.
ERK and p38 MAPK-Activated Protein Kinases: a Family of Protein Kinases with Diverse Biological Functions
Authors: Roux Philippe P. , Blenis John
Published: June 2004

Conserved signaling pathways that activate the mitogen-activated
protein kinases (MAPKs) are involved in relaying extracellular
stimulations to intracellular responses. The MAPKs
coordinately regulate cell proliferation, differentiation, motility,
and survival, which are functions also known to be mediated by members
of a growing family of MAPK-activated protein kinases (MKs;
formerly known as MAPKAP kinases). The MKs are related
serine/threonine kinases that respond to mitogenic and stress stimuli
through proline-directed
phosphorylation and activation of
the kinase domain by extracellular signal-regulated kinases 1 and 2 and
p38 MAPKs. There are currently 11 vertebrate MKs in five
subfamilies based on primary sequence homology: the ribosomal S6
kinases, the mitogen- and stress-activated kinases, the
MAPK-interacting kinases, MAPK-activated protein
kinases 2 and 3, and MK5. In the last 5 years, several MK substrates
have been identified, which has helped tremendously to identify the
biological role of the members of this family. Together with data from
the study of MK-knockout mice, the identities of the MK substrates
indicate that they play important roles in diverse biological
processes, including mRNA translation, cell proliferation and
survival, and the nuclear genomic response to mitogens and cellular
stresses. In this article, we review the existing data on the MKs and
discuss their physiological functions based on recent
discoveries.
Microbial Responses to Microgravity and Other Low-Shear Environments
Authors: Nickerson Cheryl A. , Ott C. Mark , Wilson James W. , Ramamurthy Rajee , Pierson Duane L.
Published: June 2004

Microbial adaptation to environmental stimuli is essential for survival. While several of these stimuli have been studied in detail, recent studies have demonstrated an important role for a novel environmental parameter in which microgravity and the low fluid shear dynamics associated with microgravity globally regulate microbial gene expression, physiology, and pathogenesis. In addition to analyzing fundamental questions about microbial responses to spaceflight, these studies have demonstrated important applications for microbial responses to a ground-based, low-shear stress environment similar to that encountered during spaceflight. Moreover, the low-shear growth environment sensed by microbes during microgravity of spaceflight and during ground-based microgravity analogue culture is relevant to those encountered during their natural life cycles on Earth. While no mechanism has been clearly defined to explain how the mechanical force of fluid shear transmits intracellular signals to microbial cells at the molecular level, the fact that cross talk exists between microbial signal transduction systems holds intriguing possibilities that future studies might reveal common mechanotransduction themes between these systems and those used to sense and respond to low-shear stress and changes in gravitation forces. The study of microbial mechanotransduction may identify common conserved mechanisms used by cells to perceive changes in mechanical and/or physical forces, and it has the potential to provide valuable insight for understanding mechanosensing mechanisms in higher organisms. This review summarizes recent and future research trends aimed at understanding the dynamic effects of changes in the mechanical forces that occur in microgravity and other low-shear environments on a wide variety of important microbial parameters.
Pathogenesis of Human Papillomaviruses in Differentiating Epithelia
Authors: Longworth Michelle S. , Laimins Laimonis A.
Published: June 2004

Human papillomaviruses (HPV) are the etiological agents of cervical and other anogenital malignancies. Over 100 different types of HPVs have been identified to date, and all target epithelial tissues for infection. One-third of HPV types specifically infect the genital tract, and a subset of these are the causative agents of anogenital cancers. Other HPV types that infect the genital tract induce benign hyperproliferative lesions or genital warts. The productive life cycle of HPVs is linked to epithelial differentiation. Papillomaviruses are thought to infect cells in the basal layer of stratified epithelia and establish their genomes as multicopy nuclear episomes. In these cells, viral DNA is replicated along with cellular chromosomes. Following cell division, one of the daughter cells migrates away from the basal layer and undergoes differentiation. In highly differentiated suprabasal cells, vegetative viral replication and late-gene expression are activated, resulting in the generation of progeny virions. Since virion production is restricted to differentiated cells, infected basal cells can persist for up to several decades or until the immune system clears the infection. The E6 and E7 genes encode viral oncoproteins that target Rb and p53, respectively. During the viral life cycle, these proteins facilitate stable maintenance of episomes and stimulate differentiated cells to reenter the S phase. The E1 and E2 proteins act as origin recognition factors as well as regulators of early viral transcription. The functions of the E5 and E1^E4 proteins are still largely unknown, but these proteins have been implicated in modulating late viral functions. The L1 and L2 proteins form icosahedral capsids for progeny virion generation. The characterization of the cellular targets of these viral proteins and the mechanisms regulating the differentiation-dependent viral life cycle remain active areas for the study of these important human pathogens.
Cation transport by the neuronal K+-Cl− cotransporter KCC2: thermodynamics and kinetics of alternate transport modes
Authors: Williams Jeffery R. , Payne John A.
Published: June 2004

Both Cs+ and NH4+ alter neuronal Cl− homeostasis, yet the mechanisms have not been clearly elucidated. We hypothesized that these two cations altered the operation of the neuronal K+-Cl− cotransporter (KCC2). Using exogenously expressed KCC2 protein, we first examined the interaction of cations at the transport site of KCC2 by monitoring furosemide-sensitive 86Rb+ influx as a function of external Rb+ concentration at different fixed external cation concentrations (Na+, Li+, K+, Cs+, and NH4+). Neither Na+ nor Li+ affected furosemide-sensitive 86Rb+ influx, indicating their inability to interact at the cation translocation site of KCC2. As expected for an enzyme that accepts Rb+ and K+ as alternate substrates, K+ was a competitive inhibitor of Rb+ transport by KCC2. Like K+, both Cs+ and NH4+ behaved as competitive inhibitors of Rb+ transport by KCC2, indicating their potential as transport substrates. Using ion chromatography to measure unidirectional Rb+ and Cs+ influxes, we determined that although KCC2 was capable of transporting Cs+, it did so with a lower apparent affinity and maximal velocity compared with Rb+. To assess NH4+ transport by KCC2, we monitored intracellular pH (pHi) with a pH-sensitive fluorescent dye after an NH4+-induced alkaline load. Cells expressing KCC2 protein recovered pHi much more rapidly than untransfected cells, indicating that KCC2 can mediate net NH4+ uptake. Consistent with KCC2-mediated NH4+ transport, pHi recovery in KCC2-expressing cells could be inhibited by furosemide (200 μM) or removal of external [Cl−]. Thermodynamic and kinetic considerations of KCC2 operating in alternate transport modes can explain altered neuronal Cl− homeostasis in the presence of Cs+ and NH4+.
Cytoskeletal mechanics in adherent human airway smooth muscle cells: probe specificity and scaling of protein-protein dynamics
Authors: Puig-de-Morales Marina , Millet Emil , Fabry Ben , Navajas Daniel , Wang Ning , Butler James P. , Fredberg Jeffrey J.
Published: June 2004

We probed elastic and loss moduli in the adherent human airway smooth muscle cell through a variety of receptor systems, each serving as a different molecular window on cytoskeletal dynamics. Coated magnetic microbeads were attached to the cell surface via coating-receptor binding. A panel of bead coatings was investigated: a peptide containing the sequence RGD, vitronectin, urokinase, activating antibody against β1-integrin, nonactivating antibody against β1-integrin, blocking antibody against β1-integrin, antibody against β1-integrin, and acetylated low-density lipoprotein. An oscillatory mechanical torque was applied to the bead, and resulting lateral displacements were measured at baseline, after actin disruption by cytochalasin D, or after contractile activation by histamine. As expected, mechanical moduli depended strongly on bead type and bead coating, differing at the extremes by as much as two orders of magnitude. In every case, however, elastic and loss moduli increased with frequency f as a weak power law, f  x−1. Moreover, with few exceptions, data could be scaled such that elastic and frictional responses depended solely on the power law exponent x. Taken together, these data suggest that power law behavior represents a generic feature of underlying protein-protein dynamics.
Novel effect of NF-κB activation: carbonylation and nitration injury to cytoskeleton and disruption of monolayer barrier in intestinal epithelium
Authors: Banan A. , Zhang L. J. , Shaikh M. , Fields J. Z. , Farhadi A. , Keshavarzian A.
Published: June 2004

Using monolayers of intestinal cells, we reported that upregulation of inducible nitric oxide synthase (iNOS) is required for oxidative injury and that activation of NF-κB is key to cytoskeletal instability. In the present study, we hypothesized that NF-κB activation is crucial to oxidant-induced iNOS upregulation and its injurious consequences: cytoskeletal oxidation and nitration and monolayer dysfunction. Wild-type (WT) cells were pretreated with inhibitors of NF-κB, with or without exposure to oxidant (H2O2). Other cells were transfected with an IκBα mutant (an inhibitor of NF-κB). Relative to WT cells exposed to vehicle, oxidant exposure caused increases in IκBα instability, NF-κB subunit activation, iNOS-related activity (NO, oxidative stress, tubulin nitration), microtubule disassembly and instability (increased monomeric and decreased polymeric tubulin), and monolayer disruption. Monolayers pretreated with NF-κB inhibitors (MG-132, lactacystin) were protected against oxidation, showing decreases in all measures of the NF-κB → iNOS → NO pathway. Dominant mutant stabilization of IκBα to inactivate NF-κB suppressed all measures of the iNOS/NO upregulation while protecting monolayers against oxidant insult. In these mutants, we found prevention of tubulin nitration and oxidation and enhancement of cytoskeletal and monolayer stability. We concluded that 1) NF-κB is required for oxidant-induced iNOS upregulation and for the consequent nitration and oxidation of cytoskeleton; 2) NF-κB activation causes cytoskeletal injury following upregulation of NO-driven processes; and 3) the molecular event underlying the destabilizing effects of NF-κB appears to be increases in carbonylation and nitrotyrosination of the subunit components of cytoskeleton. The ability to promote NO overproduction and cytoskeletal nitration/oxidation is a novel mechanism not previously attributed to NF-κB in cells.
Human cytomegalovirus-induced host cell enlargement is iron dependent
Authors: Crowe William E. , Maglova Lilia M. , Ponka Prem , Russell John M.
Published: June 2004

A hallmark of human cytomegalovirus (HCMV) infection is the characteristic enlargement of the host cells (i.e., cytomegaly). Because iron (Fe) is required for cell growth and Fe chelators inhibit viral replication, we investigated the effects of HCMV infection on Fe homeostasis in MRC-5 fibroblasts. Using the metallosensitive fluorophore calcein and the Fe chelator salicylaldehyde isonicotinoyl hydrazone (SIH), the labile iron pool (LIP) in mock-infected cells was determined to be 1.04 ± 0.05 μM. Twenty-four hours postinfection (hpi), the size of the LIP had nearly doubled. Because cytomegaly occurs between 24 and 96 hpi, access to this larger LIP could be expected to facilitate enlargement to ∼375% of the initial cell size. The ability of Fe chelation by 100 μM SIH to limit enlargement to ∼180% confirms that the LIP plays a major role in cytomegaly. The effect of SIH chelation on the mitochondrial membrane potential (ΔΨM) and morphology was studied using the mitochondrial voltage-sensitive dye JC-1. The mitochondria in mock-infected cells were heterogeneous with a broad distribution of ΔΨM and were threadlike. In contrast, the mitochondria of HCMV-infected cells had a more depolarized ΔΨM distributed over a narrow range and were grainlike in appearance. However, the HCMV-induced alteration in ΔΨM was not affected by SIH chelation. We conclude that the development of cytomegaly is inhibited by Fe chelation and may be facilitated by an HCMV-induced increase in the LIP.
Estrogens sensitize anterior pituitary gland to apoptosis
Authors: Pisera D. , Candolfi M. , Navarra S. , Ferraris J. , Zaldivar V. , Jaita G. , Castro M. G. , Seilicovich A.
Published: June 2004

Tissue homeostasis results from a balance between cell proliferation and cell death by apoptosis. Estradiol affects proliferation as well as apoptosis in hormone-dependent tissues. In the present study, we investigated the apoptotic response of the anterior pituitary gland to lipopolysaccharide (LPS) in cycling female rats, and the influence of estradiol in this response in ovariectomized (OVX) rats. The OVX rats were chronically estrogenized with implanted Silastic capsules containing 1 mg of 17β-estradiol (E2). Cycling or OVX and E2-treated rats were injected with LPS (250 μg/rat ip). Apoptosis was determined by the terminal deoxynucleotidyl-mediated dUTP nick-end labeling (TUNEL) method in sections of the anterior pituitary gland and spleen. Chronic estrogenization induced apoptosis in the anterior pituitary gland. Acute endotoxemia triggered apoptosis of cells in the anterior pituitary gland of E2-treated rats but not of OVX rats. No differences were observed in the apoptotic response to LPS in spleen between OVX and E2-treated rats. The apoptotic response of the anterior pituitary to LPS was variable along the estrous cycle, being higher at proestrus than at estrus or diestrus I. Approximately 75% of the apoptotic cells were identified as lactotropes by immunofluorescence. In conclusion, our results indicate that estradiol induces apoptosis and enables the proapoptotic action of LPS in the anterior pituitary gland. Also, our study suggests that estrogens may be involved in anterior pituitary cell renewal during the estrous cycle, sensitizing lactotropes to proapoptotic stimuli.
MEK inhibitors impair insulin-stimulated glucose uptake in 3T3-L1 adipocytes
Authors: Harmon Anne W. , Paul David S. , Patel Yashomati M.
Published: June 2004

In 3T3-L1 adipocytes, insulin activates three major signaling cascades, the phosphoinositide 3-kinase (PI3K) pathway, the Cbl pathway, and the mitogen-activated protein kinase (MAPK) pathway. Although PI3K and Cbl mediate insulin-stimulated glucose uptake by promoting the translocation of the insulin-responsive glucose transporter (GLUT4) to the plasma membrane, the MAPK pathway does not have an established role in insulin-stimulated glucose uptake. We demonstrate in this report that PI3K inhibitors also inhibit the MAPK pathway. To investigate the role of the MAPK pathway separately from that of the PI3K pathway in insulin-stimulated glucose uptake, we used two specific inhibitors of MAPK kinase (MEK) activity, PD-98059 and U-0126, which reduced insulin-stimulated glucose uptake by ∼33 and 50%, respectively. Neither MEK inhibitor affected the activation of Akt or PKCζ/λ, downstream signaling molecules in the PI3K pathway. Inhibition of MEK with U-0126 did not prevent GLUT4 from translocating to the plasma membrane, nor did it inhibit the subsequent docking and fusion of GLUT4- myc with the plasma membrane. MEK inhibitors affected glucose transport mediated by GLUT4 but not GLUT1. Importantly, the presence of MEK inhibitors only at the time of the transport assay markedly impaired both insulin-stimulated glucose uptake and MAPK signaling. Conversely, removal of MEK inhibitors before the transport assay restored glucose uptake and MAPK signaling. Collectively, our studies suggest a possible role for MEK in the activation of GLUT4.