bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermoprotection of
Bacillus subtilis
by Exogenously Provided Glycine Betaine and Structurally Related Compatible Solutes: Involvement of Opu Transporters
Authors: Holtmann Gudrun , Bremer Erhard
Published: March 2004

Bacillus subtilis
possesses five osmotically regulated transporters (Opu) for the uptake of various compatible solutes for osmoprotective purposes. We have now found that compatible solutes also function as thermoprotectants for
B. subtilis
. Low concentrations of glycine betaine enhanced the growth of the
B. subtilis
wild-type strain JH642 at its maximal growth temperature (52°C) but did not allow an extension of the upper growth limit. A similar enhancement in the growth of
B. subtilis
was also observed by the addition of several other compatible solutes that are structurally related to glycine betaine or by the addition of proline. Each of these compatible solutes was taken up under heat stress by the cell through the same Opu transporters that are used for their acquisition under osmostress conditions. Northern blot analysis revealed a moderate increase in transcription of the structural genes for each of the Opu transport systems in cells that were propagated at 52°C. In contrast, the uptake level of radiolabeled glycine betaine was very low under high-temperature growth conditions but nevertheless allowed the buildup of an intracellular glycine betaine pool comparable to that found in cells grown at 37°C in the absence of salt stress. Although exogenously added glutamate has only a limited osmoprotective potential for
B. subtilis
, it was found to be a very effective thermoprotectant. Collectively, our data demonstrate thermoprotection by a variety of compatible solutes in
B. subtilis
, thus ascribing a new physiological function for this class of compounds in this microorganism and broadening the physiological role of the known osmoprotectant uptake systems (Opu).
Role of
murE
in the Expression of β-Lactam Antibiotic Resistance in
Staphylococcus aureus
Authors: Gardete S. , Ludovice A. M. , Sobral R. G. , Filipe S. R. , de Lencastre H. , Tomasz A.
Published: March 2004

It was shown earlier that Tn
551
inserted into the C-terminal region of
murE
of parental methicillin-resistant
Staphylococcus aureus
strain COL causes a drastic reduction in methicillin resistance, accompanied by accumulation of UDP-MurNAc dipeptide in the cell wall precursor pool and incorporation of these abnormal muropeptides into the peptidoglycan of the mutant. Methicillin resistance was recovered in a suppressor mutant. The
murE
gene of the same strain was then put under the control of the isopropyl-β-
d
-thiogalactopyranoside (IPTG)-inducible promoter P

spac

. Bacteria grown in the presence of suboptimal concentrations of IPTG accumulated UDP-MurNAc dipeptide in the cell wall precursor pool. Both growth rates and methicillin resistance levels (but not resistance to other antibiotics) were a function of the IPTG concentration. Northern analysis showed a gradual increase in the transcription of
murE
and also in the transcription of
pbpB
and
mecA
, parallel with the increasing concentrations of IPTG in the medium. A similar increase in the transcription of
pbpB
and
mecA
, the structural genes of penicillin-binding protein 2 (PBP2) and PBP2A, was also detected in the suppressor mutant. The expression of these two proteins, which are known to play critical roles in the mechanism of staphylococcal methicillin resistance, appears to be—directly or indirectly—under the control of the
murE
gene. Our data suggest that the drastic reduction of the methicillin MIC seen in the
murE
mutant may be caused by the insufficient cellular amounts of these two PBPs.
A −1 Ribosomal Frameshift in the Transcript That Encodes the Major Head Protein of Bacteriophage A2 Mediates Biosynthesis of a Second Essential Component of the Capsid
Authors: García Pilar , Rodríguez Isabel , Suárez Juan E.
Published: March 2004

The two major capsid proteins ofLactobacillusbacteriophage A2 share their amino termini. The smaller of these (gp5A) results from translation oforf5and proteolytic processing after residue 123. The larger form (gp5B) originates through a −1 ribosomal frameshift at the penultimate codon oforf5mRNA, resulting in a product that is 85 amino acids longer than gp5A. Frameshifting needs twocis-acting elements: a slippery region with the sequence C CCA AAA (0 frame), and a stem-loop that begins 9 nucleotides after the end of the slippery sequence. Mutations introduced in the slippery sequence suppress the frameshift. Similarly, deletion of the second half of the stem-loop results in drastic reduction of frameshifting. Both gp5A and gp5B appear to be essential for phage viability, since lysogens harboring prophages that produce only one or the other protein become lysed upon induction with mitomycin C, though no viable phage progeny are observed.
Light Control ofhliATranscription and Transcript Stability in the CyanobacteriumSynechococcus elongatusStrain PCC 7942
Authors: Salem Kavitha , van Waasbergen Lorraine G.
Published: March 2004

The high-light-inducible proteins (HLIPs) of cyanobacteria are polypeptides involved in protecting the cells from high-intensity light (HL). ThehliAgene encoding the HLIP fromSynechococcus elongatusstrain PCC 7942 is expressed in response to HL or low-intensity blue or UV-A light. In this study, we explore via Northern analysis details of the transcriptional regulation and transcript stability of thehliAgene under various light conditions. Transcript levels of thehliAgene increased dramatically upon a shift to HL or UV-A light to similar levels, followed by a rapid decrease in UV-A light, but not in HL, consistent with blue/UV-A light involvement in early stages of HL-mediated expression. A 3-min pulse of low-intensity UV-A light was enough to triggerhliAmRNA accumulation, indicating that a blue/UV-A photoreceptor is involved in upregulation of the gene. Low-intensity red light was found to cause a slight, transient increase in transcript levels (raising the possibility of red-light photoreceptor involvement), while light of other qualities had no apparent effect. No evidence was found for wavelength-specific attenuation ofhliAtranscript levels induced by HL or UV-A light. Transcript decay was slowed somewhat in darkness, and when photosynthetic electron transport was inhibited by darkness or treatment with DCMU, there appeared a smaller mRNA species that may represent a decay intermediate that accumulates when mRNA decay is slowed. Evidence suggests that upregulation ofhliAby light is primarily a transcriptional response but conditions that cause ribosomes to stall on the transcript (e.g., a shift to darkness) can help stabilizehliAmRNA and affect expression levels.
Phospho-
N
-Acetyl-Muramyl-Pentapeptide Translocase from
Escherichia coli
: Catalytic Role of Conserved Aspartic Acid Residues
Authors: Lloyd Adrian J. , Brandish Philip E. , Gilbey Andrea M. , Bugg Timothy D. H.
Published: March 2004

Phospho-
N
-acetyl-muramyl-pentapeptide translocase (translocase 1) catalyzes the first of a sequence of lipid-linked steps that ultimately assemble the peptidoglycan layer of the bacterial cell wall. This essential enzyme is the target of several natural product antibiotics and has recently been the focus of antimicrobial drug discovery programs. The catalytic mechanism of translocase 1 is believed to proceed via a covalent intermediate formed between phospho-
N
-acetyl-muramyl-pentapeptide and a nucleophilic amino acid residue. Amino acid sequence alignments of the translocase 1 family and members of the related transmembrane phosphosugar transferase superfamily revealed only three conserved residues that possess nucleophilic side chains: the aspartic acid residues D115, D116, and D267. Here we report the expression and partial purification of
Escherichia coli
translocase 1 as a C-terminal hexahistidine (C-His
6
) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His
6
fusions. Enzymatic analysis established that all three mutations eliminated translocase 1 activity, and this finding verified the essential role of these residues. By analogy with the structural environment of the double aspartate motif found in prenyl transferases, we propose a model whereby D115 and D116 chelate a magnesium ion that coordinates with the pyrophosphate bridge of the UDP-
N
-acetyl-muramyl-pentapeptide substrate and in which D267 therefore fulfills the role of the translocase 1 active-site nucleophile.
In-Depth Profiling of Lysine-Producing
Corynebacterium glutamicum
by Combined Analysis of the Transcriptome, Metabolome, and Fluxome
Authors: Krömer Jens Olaf , Sorgenfrei Oliver , Klopprogge Kai , Heinzle Elmar , Wittmann Christoph
Published: March 2004

An in-depth analysis of the intracellular metabolite concentrations, metabolic fluxes, and gene expression (metabolome, fluxome, and transcriptome, respectively) of lysine-producing
Corynebacterium glutamicum
ATCC 13287 was performed at different stages of batch culture and revealed distinct phases of growth and lysine production. For this purpose,
13
C flux analysis with gas chromatography-mass spectrometry-labeling measurement of free intracellular amino acids, metabolite balancing, and isotopomer modeling were combined with expression profiling via DNA microarrays and with intracellular metabolite quantification. The phase shift from growth to lysine production was accompanied by a decrease in glucose uptake flux, the redirection of flux from the tricarboxylic acid (TCA) cycle towards anaplerotic carboxylation and lysine biosynthesis, transient dynamics of intracellular metabolite pools, such as an increase of lysine up to 40 mM prior to its excretion, and complex changes in the expression of genes for central metabolism. The integrated approach was valuable for the identification of correlations between gene expression and in vivo activity for numerous enzymes. The glucose uptake flux closely corresponded to the expression of glucose phosphotransferase genes. A correlation between flux and expression was also observed for glucose-6-phosphate dehydrogenase, transaldolase, and transketolase and for most TCA cycle genes. In contrast, cytoplasmic malate dehydrogenase expression increased despite a reduction of the TCA cycle flux, probably related to its contribution to NADH regeneration under conditions of reduced growth. Most genes for lysine biosynthesis showed a constant expression level, despite a marked change of the metabolic flux, indicating that they are strongly regulated at the metabolic level. Glyoxylate cycle genes were continuously expressed, but the pathway exhibited in vivo activity only in the later stage. The most pronounced changes in gene expression during cultivation were found for enzymes at entry points into glycolysis, the pentose phosphate pathway, the TCA cycle, and lysine biosynthesis, indicating that these might be of special importance for transcriptional control in
C. glutamicum
.
Roles of Genes 44, 50, and 51 in Regulating Gene Expression and Host Takeover during Infection of
Bacillus subtilis
by Bacteriophage SPO1
Authors: Sampath Aruna , Stewart Charles R.
Published: March 2004

We show that the products of SPO1 genes 44, 50, and 51 are required for the normal transition from early to middle gene expression during infection of
Bacillus subtilis
by bacteriophage SPO1; that they are also required for control of the shutoff of host DNA, RNA, and protein synthesis; and that their effects on host shutoff could be accounted for by their effects on the regulation of gene expression. These three gene products had four distinguishable effects in regulating SPO1 gene expression: (i) gp44-50-51 acted to restrain expression of all SPO1 genes tested, (ii) gp44 and/or gp50-51 caused additional specific repression of immediate-early genes, (iii) gp44 and/or gp50-51 stimulated expression of middle genes, and (iv) gp44 and/or gp50-51 stimulated expression of some delayed-early genes. Shutoff of immediate-early gene expression also required the activity of gp28, the middle-gene-specific sigma factor. Shutoff of host RNA and protein synthesis was accelerated by either the 44
−
single mutant or the 50
−
51
−
double mutant and more so by the 44
−
50
−
51
−
triple mutant. Shutoff of host DNA synthesis was accelerated by the mutants early in infection but delayed by the 44
−
50
−
51
−
triple mutant at later times. Although gp50 is a very small protein, consisting almost entirely of an apparent membrane-spanning domain, it contributed significantly to each activity tested. We identify SPO1 genes 41 to 51 and 53 to 60 as immediate-early genes; genes 27, 28, and 37 to 40 as delayed-early genes; and gene 52 as a middle gene.
Cloning and Sequence Analysis of Genes Encoding
Staphylococcus hyicu
s Exfoliative Toxin Types A, B, C, and D
Authors: Ahrens Peter , Andresen Lars Ole
Published: March 2004

Exfoliative toxins produced by certain strains of
Staphylococcus hyicus
mediate exudative epidermitis in pigs. In this study the genes coding for four different exfoliative toxin from
S. hyicus
(ExhA, ExhB, ExhC, and ExhD) were cloned and sequenced. The coding sequence of the four toxin genes ranged from 816 to 834 bp. The amino acid sequences of these four toxins were homologous to the earlier described exfoliative toxins SHETB from
S. hyicus
and ETA, ETB, and ETD from
Staphylococcus aureus
. The homology between the
S. hyicus
toxins was at the same level as the homology to the exfoliative toxins from
S. aureus
. The toxins showed similarity to serine proteases, including preservation of the catalytic tract in ExhA, ExhB, and ExhC. However, in ExhD, Asp in the putative catalytic tract was replaced with Glu. The recombinant toxins could be expressed in
Escherichia coli
, and three of the four toxins were recognized by monoclonal antibodies raised against native exfoliative toxins.
Quorum Sensing in
Staphylococcus aureus
Biofilms
Authors: Yarwood Jeremy M. , Bartels Douglas J. , Volper Esther M. , Greenberg E. Peter
Published: March 2004

Several serious diseases are caused by biofilm-associated
Staphylococcus aureus
, infections in which the accessory gene regulator (
agr
) quorum-sensing system is thought to play an important role. We studied the contribution of
agr
to biofilm development, and we examined
agr
-dependent transcription in biofilms. Under some conditions, disruption of
agr
expression had no discernible influence on biofilm formation, while under others it either inhibited or enhanced biofilm formation. Under those conditions where
agr
expression enhanced biofilm formation, biofilms of an
agr
signaling mutant were particularly sensitive to rifampin but not to oxacillin. Time lapse confocal scanning laser microscopy showed that, similar to the expression of an
agr
-independent fluorescent reporter, biofilm expression of an
agr
-dependent reporter was in patches within cell clusters and oscillated with time. In some cases, loss of fluorescence appeared to coincide with detachment of cells from the biofilm. Our studies indicate that the role of
agr
expression in biofilm development and behavior depends on environmental conditions. We also suggest that detachment of cells expressing
agr
from biofilms may have important clinical implications.
Isolation and Characterization of β-Ketoacyl-Acyl Carrier Protein Reductase (
fabG
) Mutants of
Escherichia coli
and
Salmonella enterica
Serovar Typhimurium
Authors: Lai Chiou-Yan , Cronan John E.
Published: March 2004

FabG, β-ketoacyl-acyl carrier protein (ACP) reductase, performs the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products, the first reductive step in the elongation cycle of fatty acid biosynthesis. We report the first documented
fabG
mutants and their characterization. By chemical mutagenesis followed by a tritium suicide procedure, we obtained three conditionally lethal temperature-sensitive
fabG
mutants. The
Escherichia coli
[
fabG
(Ts)] mutant contains two point mutations: A154T and E233K. The β-ketoacyl-ACP reductase activity of this mutant was extremely thermolabile, and the rate of fatty acid synthesis measured in vivo was inhibited upon shift to the nonpermissive temperature. Moreover, synthesis of the acyl-ACP intermediates of the pathway was inhibited upon shift of mutant cultures to the nonpermissive temperature, indicating blockage of the synthetic cycle. Similar results were observed for in vitro fatty acid synthesis. Complementation analysis revealed that only the E233K mutation was required to give the temperature-sensitive growth phenotype. In the two
Salmonella enterica
serovar Typhimurium
fabG
(Ts) mutants one strain had a single point mutation, S224F, whereas the second strain contained two mutations (M125I and A223T). All of the altered residues of the FabG mutant proteins are located on or near the twofold axes of symmetry at the dimer interfaces in this homotetrameric protein, suggesting that the quaternary structures of the mutant FabG proteins may be disrupted at the nonpermissive temperature.
The
eutD
Gene of
Salmonella enterica
Encodes a Protein with Phosphotransacetylase Enzyme Activity
Authors: Brinsmade Shaun R. , Escalante-Semerena Jorge C.
Published: March 2004

The EutD protein of
Salmonella enterica
is homologous to the catalytic domain of the phosphotransacetylase (Pta) enzyme. The Pta-like activity level of the EutD enzyme compared favorably to that of other Pta enzymes. High-pressure liquid chromatography and mass spectrometry verified that acetyl-coenzyme A was the product of the reaction. The EutD protein restored growth of an
S. enterica pta
strain on acetate as the source of carbon and energy.
Analysis of an Agmatine Deiminase Gene Cluster in
Streptococcus mutans
UA159
Authors: Griswold Ann R. , Chen Yi-Ywan M. , Burne Robert A.
Published: March 2004

An operon encoding enzymes of the agmatine deiminase system (AgDS) has been identified in the cariogenic bacterium
Streptococcus mutans
UA159. The AgDS is regulated by agmatine induction and carbohydrate catabolite repression. Ammonia is produced from agmatine at low pH, suggesting that the AgDS could augment acid tolerance.
Transcriptional Phase Variation at the
flhB
Gene of
Pseudomonas putida
DOT-T1E Is Involved in Response to Environmental Changes and Suggests the Participation of the Flagellar Export System in Solvent Tolerance
Authors: Segura Ana , Hurtado Ana , Duque Estrella , Ramos Juan L.
Published: March 2004

Frameshift mutations in a poly(G) track at the
flhB
gene of
Pseudomonas putida
DOT-T1E are responsible for the diminished swimming of this strain on semisolid medium, which contrasts with the high swimming ability of
P. putida
KT2440, which does not exhibit a poly(G) track at the
flhB
gene. We previously showed that a mutant lacking FlhB was more sensitive to solvents than the wild-type strain (Segura et al., J. Bacteriol., 183:4127-4133, 2001). In this study, we show that swimming ability correlates with solvent tolerance in
P. putida
DOT-T1E, so that growth conditions favoring a functional
flhB
gene (growth on semisolid medium) resulted in increased innate tolerance to a sudden toluene shock.
Functional and molecular identification of intermediate-conductance Ca2+-activated K+ channels in breast cancer cells: association with cell cycle progression
Authors: Ouadid-Ahidouch Halima , Roudbaraki Morad , Delcourt Philippe , Ahidouch Ahmed , Joury Nathalie , Prevarskaya Natalia
Published: March 2004

We have previously reported that the hEAG K+ channels are responsible for the potential membrane hyperpolarization that induces human breast cancer cell progression into the G1 phase of the cell cycle. In the present study, we evaluate the role and functional expression of the intermediate-conductance Ca2+-activated K+ channel, hIK1-like, in controlling cell cycle progression. Our results demonstrate that hIK1 current density increased in cells synchronized at the end of the G1 or S phase compared with those in the early G1 phase. This increased current density paralleled the enhancement in hIK1 mRNA levels and the highly negative membrane potential. Furthermore, in cells synchronized at the end of G1 or S phases, basal cytosolic Ca2+ concentration ([Ca2+]i) was also higher than in cells arrested in early G1. Blocking hIK1 channels with a specific blocker, clotrimazole, induced both membrane potential depolarization and a decrease in the [Ca2+]i in cells arrested at the end of G1 and S phases but not in cells arrested early in the G1 phase. Blocking hIK1 with clotrimazole also induced cell proliferation inhibition but to a lesser degree than blocking hEAG with astemizole. The two drugs were essentially additive, inhibiting MCF-7 cell proliferation by 82% and arresting >90% of cells in the G1 phase. Thus, although the progression of MCF-7 cells through the early G1 phase is dependent on the activation of hEAG K+ channels, when it comes to G1 and checkpoint G1/S transition, the membrane potential appears to be primarily dependent on the hIK1-activity level.
Acute metabolic acidosis inhibits muscle protein synthesis in rats
Authors: Caso Giuseppe , Garlick Barbara A. , Casella George A. , Sasvary Dawn , Garlick Peter J.
Published: March 2004

In this study, we investigated the effect of acute metabolic acidosis on tissue protein synthesis. Groups of rats were made acidotic with intragastric administration of NH4Cl (20 mmol/kg body wt every 12 h for 24 h) or given equimolar amounts of NaCl (controls). Protein synthesis in skeletal muscle and a variety of different tissues, including lymphocytes, was measured after 24 h by injection of l-[2H5]phenylalanine (150 μmol/100 g body wt, 40 moles percent). Results show that acute acidosis inhibits protein synthesis in skeletal muscle (−29% in gastrocnemius, −23% in plantaris, and −17% in soleus muscles, P < 0.01) but does not affect protein synthesis in heart, liver, gut, kidney, and spleen. Protein synthesis in lymphocytes is also reduced by acidosis (−8%, P < 0.05). In a separate experiment, protein synthesis was also measured in acidotic and control rats by a constant infusion of l-[2H5]phenylalanine (1 μmol·100 g body wt−1·h−1). The results confirm the earlier findings showing an inhibition of protein synthesis in gastrocnemius (−28%, P < 0.01) and plantaris (−19%, P < 0.01) muscles but no effect on heart and liver by acidosis. Similar results were also observed using a different model of acute metabolic acidosis, in which rats were given a cation exchange resin in the H+(acidotic) or the Na+(controls) form. In conclusion, this study demonstrates that acute metabolic acidosis for 24 h depresses protein synthesis in skeletal muscle and lymphocytes but does not alter protein synthesis in visceral tissues. Inhibition of muscle protein synthesis might be another mechanism contributing to the loss of muscle tissue observed in acidosis.
Sim1 gene dosage modulates the homeostatic feeding response to increased dietary fat in mice
Authors: Holder J. Lloyd , Zhang Ling , Kublaoui Bassil M. , DiLeone Ralph J. , Oz Orhan K. , Bair Chi Horng , Lee Ying-Hue , Zinn Andrew R.
Published: March 2004

Haploinsufficiency of the transcription factor gene Sim1 has been previously implicated in hyperphagic obesity in humans and mice. To investigate the relation between Sim1 dosage and hyperphagia, we generated sim1-knockout mice and studied their growth and feeding behavior. Heterozygous mice weaned on standard chow consumed 14% more food per day than controls and developed obesity, hyperinsulinemia, and hyperleptinemia. The sim1 heterozygous mice were also significantly longer than controls. Heterozygous animals had modestly increased feeding efficiency, suggesting reduced energy expenditure, but voluntary wheel-running activity did not differ significantly between the two groups. We studied the effect of dietary fat on the feeding behavior of heterozygous sim1 mutant mice. The tempo and severity of weight gain were much greater in animals weaned on a high-fat diet. When acutely challenged with increased dietary fat, sim1 heterozygous mice weaned on the chow diet markedly increased their food consumption and caloric intake, whereas control mice reduced the mass of food they consumed and maintained approximately isocaloric intake. In wild-type adult mice, we detected Sim1 expression in the paraventricular and supraoptic nuclei, as previously reported in neonates, as well as in the amygdala and lateral hypothalamus, all regions implicated in feeding behavior. Our results indicate that Sim1 gene dosage modulates the homeostatic feeding response to increased dietary fat and likely plays a physiological role in the regulation of energy balance.
Immunolocalization of AQP-5 in rat parotid and submandibular salivary glands after stimulation or inhibition of secretion in vivo
Authors: Gresz Veronika , Kwon Tae-Hwan , Gong Hong , Agre Peter , Steward Martin C. , King Landon S. , Nielsen Søren
Published: March 2004

In vitro studies of cultured salivary gland cells and gland slices have indicated that there may be regulated translocation of aquaporin (AQP)-5 between the apical plasma membrane and intracellular compartments of the secretory cells. However, it remains unknown whether AQP-5 in salivary glands is subject to regulated trafficking in vivo. To examine this possibility, we have investigated the subcellular localization of AQP-5 in rat parotid and submandibular glands fixed in vivo under conditions of stimulated or inhibited salivary secretion. Immunofluorescence and immunoelectron microscopy was used to determine the subcellular distribution of AQP-5 in control conditions following the stimulation of secretion with pilocarpine (a muscarinic agonist) or epinephrine (an α-adrenoceptor agonist) or during inhibition of basal secretion with atropine (a muscarinic antagonist) or phentolamine (an α-adrenoceptor antagonist). Under control conditions, >90% of AQP-5 was associated with the apical plasma membrane of acinar and intercalated duct cells, with only rare gold particles associated with intracellular membrane domains. Pilocarpine treatment dramatically increased saliva production but had no discernible effect on AQP-5 distribution. However, the increased salivary secretion was associated with luminal dilation and the appearance of a markedly punctate AQP-5 labeling pattern due to clustering of AQP-5 at the microvilli (especially evident in the parotid gland) after 10 min of drug injection. No changes in the subcellular localization of AQP-5 were seen in response to epinephrine, atropine, or phentolamine treatment compared with control tissues. Thus AQP-5 is localized predominantly in the apical plasma membrane under control conditions, and neither the onset nor the cessation of secretion is associated in vivo with any significant short-term translocation of AQP-5 between intracellular structures and the apical plasma membrane.
R-type calcium channels in myenteric neurons of guinea pig small intestine
Authors: Bian Xiaochun , Zhou Xiaoping , Galligan James J.
Published: March 2004

Currents carried by L-, N-, and P/Q-type calcium channels do not account for the total calcium current in myenteric neurons. This study identified all calcium channels expressed by guinea pig small intestinal myenteric neurons maintained in primary culture. Calcium currents were recorded using whole cell techniques. Depolarizations (holding potential = −70 mV) elicited inward currents that were blocked by CdCl2 (100 μM). Combined application of nifedipine (blocks L-type channels), Ω-conotoxin GVIA (blocks N-type channels), and Ω-agatoxin IVA (blocks P/Q-type channels) inhibited calcium currents by 56%. Subsequent addition of the R-type calcium channel antagonists, NiCl2 (50 μM) or SNX-482 (0.1 μM), abolished the residual calcium current. NiCl2 or SNX-482 alone inhibited calcium currents by 46%. The activation threshold for R-type calcium currents was −30 mV, the half-activation voltage was −5.2 ± 5 mV, and the voltage sensitivity was 17 ± 3 mV. R-type currents activated fully in 10 ms at 10 mV. R-type calcium currents inactivated in 1 s at 10 mV, and they inactivated (voltage sensitivity of 16 ± 1 mV) with a half-inactivation voltage of −76 ± 5 mV. These studies have accounted for all of the calcium channels in myenteric neurons. The data indicate that R-type calcium channels make the largest contribution to the total calcium current in myenteric neurons. The relatively positive half-activation voltage and rapid activation kinetics suggest that R-type channels could contribute to calcium entry during somal action potentials or during action potential-induced neurotransmitter release.
Tyrosine phosphatase inhibition augments collateral blood flow in a rat model of peripheral vascular disease
Authors: Carr Andrew N. , Davis Michael G. , Eby-Wilkens Elaine , Howard Brian W. , Towne Bryan A. , Dufresne Thomas E. , Peters Kevin G.
Published: March 2004

During embryonic development, the growth of blood vessels requires the coordinated activation of endothelial receptor tyrosine kinases (RTKs) such as vascular endothelial growth factor receptor-2 (VEGFR-2) and Tie-2. Similarly, in adulthood, activation of endothelial RTKs has been shown to enhance development of the collateral circulation and improve blood flow to ischemic tissues. Recent evidence suggests that RTK activation is negatively regulated by protein tyrosine phosphatases (PTPs). In this study, we used the nonselective PTP inhibitor bis(maltolato)oxovanadium IV (BMOV) to test the potential efficacy of PTP inhibition as a means to enhance endothelial RTK activation and improve collateral blood flow. In cultured endothelial cells, pretreatment with BMOV augmented VEGFR-2 and Tie-2 tyrosine phosphorylation and enhanced VEGF- and angiopoietin-1-mediated cell survival. In rat aortic ring explants, BMOV enhanced vessel sprouting, a process that can be influenced by both VEGFR-2 and Tie-2 activation. Moreover, 2 wk of BMOV treatment in a rat model of peripheral vascular disease enhanced collateral blood flow similarly to VEGF, and after 4 wk, BMOV was superior to VEGF. Taken together, these studies provide evidence that PTPs are important regulators of endothelial RTK activation and for the first time demonstrate the potential utility of phosphatase inhibition as a means to promote collateral development and enhance collateral blood flow to ischemic tissue.
Evidence for altered ETB receptor characteristics during development and progression of ventricular cardiomyocyte hypertrophy
Authors: Lee Graham R. , Bell David , Kelso Elizabeth J. , Argent Cymone C. H. , McDermott Barbara J.
Published: March 2004

The hypothesis that endothelin (ET) receptor mechanisms are altered during development and progression of left ventricular hypertrophy (LVH) in vivo was tested using spontaneously hypertensive rats (SHRs). Ventricular cardiomyocytes were isolated from SHRs before onset (8 and 12 wk) and during progression (16, 20, and 24 wk) of LVH and compared with age-matched normotensive Wistar-Kyoto (WKY) rats. PreproET-1 mRNA expression was elevated in SHR ( P < 0.05) relative to WKY cardiomyocytes at 20–24 wk. ET binding-site density was twofold greater in SHR than WKY cells at 12 wk ( P < 0.05) but normalized at 20 wk. ETB receptors were detected on SHR cardiomyocytes as early as 8 wk and their affinity increased progressively with age ( P < 0.05), whereas ETB receptors were not detected on WKY cells until 20 wk. ET-1 stimulated protein synthesis with similar maximum responses between strains (21–30%), in contrast with sarafotoxin 6c, which stimulated protein synthesis in SHR (13–20%) but not WKY cells at 12–20 wk. In SHR but not WKY cells, the ETB receptor-selective ligand A-192621 increased protein synthesis progressively with the development of LVH (15% maximum effect). In conclusion, the presence of ETB receptors (8–12 wk) coupled with functional responsiveness of SHR cells but not WKY cells to sarafotoxin 6c at 12 wk supports the involvement of ETB receptors before the onset of cardiomyocyte hypertrophy, whereas altered ETB receptor characteristics during active hypertrophy (16–24 wk) indicate that ETB receptor mechanisms may also contribute to disease progression.
O2 consumption of mechanically unloaded contractions of mouse left ventricular myocardial slices
Authors: Yamashita Daisuke , Kohzuki Hisaharu , Kitagawa Yutaka , Nakashima Tamiji , Kikuta Akio , Takaki Miyako
Published: March 2004

Left ventricular (LV) myocardial slices were isolated from murine hearts (300 μm thick) and were stimulated at 1 Hz without external load. Mean myocardial slice O2 consumption (MVo2) per minute (mMVo2) without stimulation was 0.97 ± 0.14 ml O2·min−1·100 g LV−1 and mean mMVo2 with stimulation increased to 1.80 ± 0.17 ml O2·min−1·100 g LV−1 in normal Tyrode solution. Mean ΔmVo2 (the mMVo2 with stimulation − the mMVo2 without stimulation) was 0.83 ± 0.12 ml O2·min−1·100 g LV−1. There were no differences between mean mMVo2 with and without stimulation in Ca2+-free solution. The increases in extracellular Ca2+ concentrations up to 14.4 mM did not affect the mMVo2 without stimulation but significantly increased the mMVo2 with stimulation up to 140% of control. The ΔmMVo2 significantly increased up to 190% of the control in a dose-dependent manner. In contrast, the shortening did not increase in a dose-dependent manner. Cyclopiazonic acid (CPA; 30 μM) significantly reduced the ΔmMVo2 to 0.27 ± 0.06 ml O2·min−1·100 g LV−1 (35% of control). The combination of 5 mM 2,3-butanedione monoxime (BDM) and 30 μM CPA did not further decrease ΔmMVo2. Although BDM (3–5 mM) decreased the ΔmMVo2 by 28–30% of control in a dose-independent manner, 3–5 mM BDM decreased shortening in a dose-dependent manner. Our results indicate that the ΔmMVo2 of mouse LV slices during shortening under mechanically unloaded conditions consists of energy expenditure for total Ca2+ handling during excitation-contraction coupling, basal metabolism, but no residual cross-bridge cycling.
Branchial membrane-associated carbonic anhydrase activity maintains CO2excretion in severely anemic dogfish
Authors: Gilmour K. M. , Perry S. F.
Published: March 2004

Plasma CO2reactions in Pacific spiny dogfish ( Squalus acanthias) have access to plasma and gill membrane-associated carbonic anhydrase (CA). Acute severe experimental anemia and selective CA inhibitors were used to investigate the role of extracellular CA in CO2excretion. Anemia was induced by blood withdrawal coupled to volume replacement with saline. Lowering hematocrit from 14.2 ± 0.4% (mean ± SE; N = 31) to 5.2 ± 0.1% ( N = 31) had no significant impact on arterial or venous CO2tensions (PaCO2and PvCO2, respectively) over the subsequent 2 h. Pco2was maintained despite the reduction in red cell number and a significant 32% increase in cardiac output (V̇b), both of which have been found to cause PaCO2increases in teleost fish. By contrast, treatment of anemic dogfish with the CA inhibitors benzolamide (1.3 mg/kg) or F3500 (50 mg/kg), to selectively inhibit extracellular CA, elicited rapid and significant increases in PaCO2of 0.68 ± 0.17 Torr ( N = 6) and 0.53 ± 0.11 Torr ( N = 7), respectively, by 30 min after treatment. These findings provide a functional context in which extracellular CA in dogfish contributes substantially to CO2excretion. Additionally, the apparent lack of effect of V̇bchanges on Pco2suggests that, in contrast to teleost fish, CO2excretion in dogfish does not behave as a diffusion-limited system.
Putative GH pulse renewal: periventricular somatostatinergic control of an arcuate-nuclear somatostatin and GH-releasing hormone oscillator
Authors: Farhy Leon S. , Veldhuis Johannes D.
Published: March 2004

Growth hormone (GH) pulsatility requires periventricular-nuclear somatostatin(SRIFPeV), arcuate-nuclear (ArC) GH-releasing hormone (GHRH), and systemic GH autofeedback. However, no current formalism interlinks these regulatory loci in a manner that generates self-renewable GH dynamics. The latter must include in the adult rat 1) infrequent volleys of high-amplitude GH peaks in the male, 2) frequent discrete low-amplitude GH pulses in the female, 3) disruption of the male pattern by severing SRIFPeV outflow to ArC, 4) stimulation of GHRH and GH secretion by central nervous system delivery of SRIF, 5) inhibition of GH release by central exposure to GHRH, and 6) a reboundlike burst of GHRH secretion induced by stopping peripheral infusion of SRIF. The present study validates by computer-assisted simulations a simplified ensemble formulation that predicts each of the foregoing six outcomes, wherein 1) blood-borne GH stimulates SRIFPeV secretion after a long time latency, 2) SRIFPeV inhibits both pituitary GH and ArC GHRH release, 3) ArC GHRH and SRIFArC oscillate reciprocally with brief time delay, and 4) SRIFPeV represses and disinhibits the putative GHRH-SRIFArC oscillator. According to the present analytic construction, time-delayed feedforward and feedback signaling among SRIFPeV, ArC GHRH, and SRIFArC could endow the complex physiological patterns of GH secretion in the male and female.
Acute increases in arterial blood pressure do not reduce plasma vasopressin levels stimulated by angiotensin II or hyperosmolality in rats
Authors: Stocker Sean D. , Schiltz Jennifer C. , Sved Alan F.
Published: March 2004

The present study sought to determine whether an acute increase in arterial blood pressure (ABP) reduces plasma vasopressin (VP) levels stimulated by ANG II or hyperosmolality. During an intravenous infusion of ANG II (100 ng·kg−1·min−1), attenuation of the ANG II-evoked increase in ABP with diazoxide or minoxidil did not further enhance plasma VP levels in rats. When VP secretion was stimulated by an infusion of hypertonic saline, coinfusion of the α-adrenergic agonist phenylephrine (PE) significantly increased ABP but did not reduce plasma VP levels. In fact, plasma VP levels were enhanced. The enhancement of plasma VP levels cannot be explained by a direct stimulatory action of PE, as plasma VP levels of isosmotic rats did not change during a similar infusion of PE. An infusion of endothelin-1 in hyperosmotic rats significantly raised ABP but did not reduce plasma VP levels; rather, VP levels increased as observed with PE. In α-chloralose-anesthetized rats infused with hypertonic saline, inflation of an aortic cuff to increase ABP and stimulate arterial baroreceptors did not reduce plasma VP levels. In each experiment, plasma oxytocin levels paralleled plasma VP levels. Collectively, the present findings suggest that an acute increase in ABP does not inhibit VP secretion.
Menstrual cycle phase and oral contraceptive effects on triglyceride mobilization during exercise
Authors: Casazza Gretchen A. , Jacobs Kevin A. , Suh Sang-Hoon , Miller Benjamin F. , Horning Michael A. , Brooks George A.
Published: March 2004

We examined the effects of menstrual cycle phase and oral contraceptive (OC) use on triglyceride mobilization during 90 min of rest and 60 min of leg ergometry exercise at 45 and 65% peak O2 uptake (V̇o2 peak) in eight moderately physically active, eumenorrheic women (24.8 ± 1.2 yr). Subjects were tested during the follicular phase (FP) and the luteal phase (LP) before OC use and during the inactive phase (IP) and high-dose phase (HP) after 4 complete mo of OC use. Glycerol rate of appearance (Ra), a measure of triglyceride mobilization, was determined in a 3-h postabsorptive state using a primed constant infusion of [1,1,2,3,3-2H]glycerol. Before OC use (BOC), there were no significant differences between FP and LP in any of the variables studied. Dietary composition, exercise patterns, plasma glycerol concentrations, growth hormone concentrations, and exercise respiratory exchange ratio did not change with OC use. However, 4 mo of OC use significantly ( P < 0.05) increased glycerol Ra in HP during exercise at 45% V̇o2 peak (6.2 ± 0.2, 6.5 ± 0.4, and 7.7 ± 1.1 μmol·kg−1·min−1 for BOC, IP, and HP, respectively) and in IP and HP at 65% V̇o2 peak (6.6 ± 0.1, 8.2 ± 0.6, and 8.1 ± 0.7 μmol·kg−1·min−1 for BOC, IP, and HP, respectively). Plasma cortisol concentrations were significantly higher with OC use at rest and during exercise at 45 and 65% V̇o2 peak. In summary, although fluctuations of endogenous ovarian steroids have little effect on triglyceride mobilization, the synthetic ovarian steroids found in OCs increase triglyceride mobilization and plasma cortisol concentrations in exercising women. We conclude that the hierarchy of effects of ovarian steroids and their analogs on triglyceride mobilization in exercising women is as follows: energy flux > OC use > recent carbohydrate nutrition, menstrual cycle effects.
Developmental changes in upper airway dynamics
Authors: Marcus Carole L. , Fernandes Do Prado Lucila B. , Lutz Janita , Katz Eliot S. , Black Cheryl A. , Galster Patricia , Carson Kathryn A.
Published: March 2004

Normal children have a less collapsible upper airway in response to subatmospheric pressure administration (PNEG) during sleep than normal adults do, and this upper airway response appears to be modulated by the central ventilatory drive. Children have a greater ventilatory drive than adults. We, therefore, hypothesized that children have increased neuromotor activation of their pharyngeal airway during sleep compared with adults. As infants have few obstructive apneas during sleep, we hypothesized that infants would have an upper airway that was resistant to collapse. We, therefore, compared the upper airway pressure-flow (V̇) relationship during sleep between normal infants, prepubertal children, and adults. We evaluated the upper airway response to 1) intermittent, acute PNEG (infants, children, and adults), and 2) hypercapnia (children and adults). We found that adults had a more collapsible upper airway during sleep than either infants or children. The children exhibited a vigorous response to both PNEG and hypercapnia during sleep ( P < 0.01), whereas adults had no significant change. Infants had an airway that was resistant to collapse and showed a very rapid response to PNEG. We conclude that the upper airway is resistant to collapse during sleep in infants and children. Normal children have preservation of upper airway responses to PNEG and hypercapnia during sleep, whereas responses are diminished in adults. Infants appear to have a different pattern of upper airway activation than older children. We speculate that the pharyngeal airway responses present in normal children are a compensatory response for a relatively narrow upper airway.
Skeletal muscle collagen content in humans after high-force eccentric contractions
Authors: Mackey Abigail L. , Donnelly Alan E. , Turpeenniemi-Hujanen Taina , Roper Helen P.
Published: March 2004

The purpose of this study was to investigate the effects of high-force eccentric muscle contractions on collagen remodeling and on circulating levels of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in humans. Nine volunteers [5 men and 4 women, mean age 23 (SD 4) yr] each performed a bout of 100 maximum voluntary eccentric contractions of the knee extensors. Muscle biopsies were taken before exercise and on days 4 and 22 afterward. Image analysis of stained tissue sections was used to quantify endomysial collagen staining intensity. Maximum voluntary contractile isometric force was recorded preexercise and on days 1, 2, 3, 4, 8, 11, and 14 postexercise. Venipuncture blood samples were also drawn on these days for measurement of serum creatine kinase activity and concentrations of MMP-9, TIMP-1, TIMP-2, and the MMP-2/TIMP-2 complex. Maximum voluntary contractile force declined by 39 ± 23% (mean ± SD) on day 2 postexercise and recovered thereafter. Serum creatine kinase activity peaked on day 4 postexercise ( P < 0.01). Collagen type IV staining intensity increased significantly on day 22 postexercise to 126 ± 29% (mean ± SD) of preexercise values ( P < 0.05). Serum MMP-9 levels increased on day 8 postexercise ( P < 0.01), and serum TIMP-1 was also significantly elevated on days 1, 2, 3, 4, and 14 postexercise ( P < 0.05). These results suggest that a single bout of eccentric muscle contractions results in remodeling of endomysial type IV collagen, possibly via the MMP pathway.
Phosphate uptake in rat skeletal muscle is reduced during isometric contractions
Authors: Abraham Kirk A. , Terjung Ronald L.
Published: March 2004

During contractions, there is a net efflux of phosphate from skeletal muscle, likely because of an elevated intracellular inorganic phosphate (Pi) concentration. Over time, contracting muscle could incur a substantial phosphate deficit unless Piuptake rates were increased during contractions. We used the perfused rat hindquarter preparation to assess [32P]Piuptake rates in muscles at rest or over a range of energy expenditures during contractions at 0.5, 3, or 5 Hz for 30 min. Piuptake rates were reduced during contractions in a pattern that was dependent on contraction frequency and fiber type. In soleus and red gastrocnemius, [32P]Piuptake rates declined by ∼25% at 0.5 Hz and 50–60% at 3 and 5 Hz. Uptake rates in white gastrocnemius decreased by 65–75% at all three stimulation frequencies. These reductions in Piuptake are not likely confounded by changes in precursor [32P]Pispecific activity in the interstitium. In soleus and red gastrocnemius, declines in Piuptake rates were related to energy expenditure over the contraction duration. These data imply that Piuptake in skeletal muscle is acutely modulated during contractions and that decreases in Piuptake rates, in combination with expected increases in Piefflux, exacerbate the net loss of phosphate from the cell. Enhanced uptake of Pimust subsequently occur because skeletal muscle typically maintains a relatively constant total phosphate pool.
Pyruvate shuttling during rest and exercise before and after endurance training in men
Authors: Henderson Gregory C. , Horning Michael A. , Lehman Steven L. , Wolfel Eugene E. , Bergman Bryan C. , Brooks George A.
Published: March 2004

We describe the isotopic exchange of lactate and pyruvate after arm vein infusion of [3-13C]lactate in men during rest and exercise. We tested the hypothesis that working muscle (limb net lactate and pyruvate exchange) is the source of the elevated systemic lactate-to-pyruvate concentration ratio (L/P) during exercise. We also hypothesized that the isotopic equilibration between lactate and pyruvate would decrease in arterial blood as glycolytic flux, as determined by relative exercise intensity, increased. Nine men were studied at rest and during exercise before and after 9 wk of endurance training. Although during exercise arterial pyruvate concentration decreased to below rest values ( P < 0.05), pyruvate net release from working muscle was as large as lactate net release under all exercise conditions. Exogenous (arterial) lactate was the predominant origin of pyruvate released from working muscle. With no significant effect of exercise intensity or training, arterial isotopic equilibration [(IEpyruvate/IElactate)·100%, where IE is isotopic enrichment] decreased significantly ( P < 0.05) from 60 ± 3.1% at rest to an average value of 12 ± 2.7% during exercise, and there were no changes in femoral venous isotopic equilibration. These data show that 1) the isotopic equilibration between lactate and pyruvate in arterial blood decreases significantly during exercise; 2) working muscle is not solely responsible for the decreased arterial isotopic equilibration or elevated arterial L/P occurring during exercise; 3) working muscle releases similar amounts of lactate and pyruvate, the predominant source of the latter being arterial lactate; 4) pyruvate clearance from blood occurs extensively outside of working muscle; and 5) working muscle also releases alanine, but alanine release is an order of magnitude smaller than lactate or pyruvate release. These results portray the complexity of metabolic integration among diverse tissue beds in vivo.