bluuebunny/crossref_metadata_2025 huggingface dataset:
Phylogenetic Analysis of Polyketide Synthase I Domains from Soil Metagenomic Libraries Allows Selection of Promising Clones
Authors: Ginolhac Aurélien , Jarrin Cyrille , Gillet Benjamin , Robe Patrick , Pujic Petar , Tuphile Karine , Bertrand Hélène , Vogel Timothy M. , Perrière Guy , Simonet Pascal , Nalin Renaud
Published: September 2004

The metagenomic approach provides direct access to diverse unexplored genomes, especially from uncultivated bacteria in a given environment. This diversity can conceal many new biosynthetic pathways. Type I polyketide synthases (PKSI) are modular enzymes involved in the biosynthesis of many natural products of industrial interest. Among the PKSI domains, the ketosynthase domain (KS) was used to screen a large soil metagenomic library containing more than 100,000 clones to detect those containing PKS genes. Over 60,000 clones were screened, and 139 clones containing KS domains were detected. A 700-bp fragment of the KS domain was sequenced for 40 of 139 randomly chosen clones. None of the 40 protein sequences were identical to those found in public databases, and nucleic sequences were not redundant. Phylogenetic analyses were performed on the protein sequences of three metagenomic clones to select the clones which one can predict to produce new compounds. Two PKS-positive clones do not belong to any of the 23 published PKSI included in the analysis, encouraging further analyses on these two clones identified by the selection process.
Genetic Structure of a
Spodoptera frugiperda
Nucleopolyhedrovirus Population: High Prevalence of Deletion Genotypes
Authors: Simón Oihane , Williams Trevor , López-Ferber Miguel , Caballero Primitivo
Published: September 2004

A Nicaraguan field isolate (SfNIC) of
Spodoptera frugiperda
nucleopolyhedrovirus was purified by plaque assay on Sf9 cells. Nine distinct genotypes, A to I, were identified by their restriction endonuclease profiles. Variant SfNIC-B was selected as the standard because its restriction profile corresponded to that of the wild-type isolate. Physical maps were generated for each of the variants. The differences between variants and the SfNIC-B standard were confined to the region between map units 9 and 32.5. This region included PstI-G, PstI-F, PstI-L, PstI-K and EcoRI-L fragments. Eight genotypes presented a deletion in their genome compared with SfNIC-B. Occlusion body-derived virions of SfNIC-C, -D and -G accounted for 41% of plaque-purified clones. These variants were not infectious per os but retained infectivity by injection into
S. frugiperda
larvae. Median 50% lethal concentration values for the other cloned genotypes were significantly higher than that of the wild type. The variants also differed in their speed of kill. Noninfectious variants SfNIC-C and -D lacked the
pif
and
pif-2
genes. Infectivity was restored to these variants by plasmid rescue with a plasmid comprising both
pif
and
pif-2
. Transcription of an SfNIC-G gene was detected by reverse transcription-PCR in insects, but no fatal disease developed. Transcription was not detected in SfNIC-C or -D-inoculated larvae. We conclude that the SfNIC population presents high levels of genetic diversity, localized to a 17-kb region containing
pif
and
pif-2
, and that interactions among complete and deleted genotypic variants will likely influence the capacity of this virus to control insect pests.
Alkaline Anaerobic Respiration: Isolation and Characterization of a Novel Alkaliphilic and Metal-Reducing Bacterium
Authors: Ye Qi , Roh Yul , Carroll Susan L. , Blair Benjamin , Zhou Jizhong , Zhang Chuanlun L. , Fields Matthew W.
Published: September 2004

Iron-reducing enrichments were obtained from leachate ponds at the U.S. Borax Company in Boron, Calif. Based on partial small-subunit (SSU) rRNA gene sequences (approximately 500 nucleotides), six isolates shared 98.9% nucleotide identity. As a representative, the isolate QYMF was selected for further analysis. QYMF could be grown with Fe(III)-citrate, Fe(III)-EDTA, Co(III)-EDTA, or Cr(VI) as electron acceptors, and yeast extract and lactate could serve as electron donors. Growth during iron reduction occurred over the pH range of 7.5 to 11.0 (optimum, pH 9.5), a sodium chloride range of 0 to 80 g/liter (optimum, 20 g/liter), and a temperature range of 4 to 45°C (optimum, approximately 35°C), and iron precipitates were formed. QYMF was a strict anaerobe that could be grown in the presence of borax, and the cells were straight rods that produced endospores. Sodium chloride and yeast extract stimulated growth. Phylogenetic analysis of the SSU rRNA gene indicated that the bacterium was a low-G+C gram-positive microorganism and had 96 and 92% nucleotide identity with
Alkaliphilus transvaalensis
and
Alkaliphilus crotonatoxidans
, respectively. The major phospholipid fatty acids were 14:1, 16:1ω7c, and 16:0, which were different from those of other alkaliphiles but similar to those of reported iron-reducing bacteria. The results demonstrated that the isolate might represent a novel metal-reducing alkaliphilic species. The name
Alkaliphilus metalliredigens
sp. nov. is proposed. The isolation and activity of metal-reducing bacteria from borax-contaminated leachate ponds suggest that bioremediation of metal-contaminated alkaline environments may be feasible and have implications for alkaline anaerobic respiration.
Raw Cow Milk Bacterial Population Shifts Attributable to Refrigeration
Authors: Lafarge Véronique , Ogier Jean-Claude , Girard Victoria , Maladen Véronique , Leveau Jean-Yves , Gruss Alexandra , Delacroix-Buchet Agnès
Published: September 2004

We monitored the dynamic changes in the bacterial population in milk associated with refrigeration. Direct analyses of DNA by using temporal temperature gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) allowed us to make accurate species assignments for bacteria with low-GC-content (low-GC%) (<55%) and medium- or high-GC% (>55%) genomes, respectively. We examined raw milk samples before and after 24-h conservation at 4°C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database (∼150 species) that we established with DNA fragments of pure bacterial strains. Cloning and sequencing of fragments missing from the database were used to achieve complete species identification. Considerable evolution of bacterial populations occurred during conservation at 4°C. TTGE and DGGE are shown to be a powerful tool for identifying the main bacterial species of the raw milk samples and for monitoring changes in bacterial populations during conservation at 4°C. The emergence of psychrotrophic bacteria such as
Listeria
spp. or
Aeromonas hydrophila
is demonstrated.
Metabolic Primers for Detection of (Per)chlorate-Reducing Bacteria in the Environment and Phylogenetic Analysis of
cld
Gene Sequences
Authors: Bender Kelly S. , Rice Melissa R. , Fugate William H. , Coates John D. , Achenbach Laurie A.
Published: September 2004

Natural attenuation of the environmental contaminant perchlorate is a cost-effective alternative to current removal methods. The success of natural perchlorate remediation is dependent on the presence and activity of dissimilatory (per)chlorate-reducing bacteria (DPRB) within a target site. To detect DPRB in the environment, two degenerate primer sets targeting the chlorite dismutase (
cld
) gene were developed and optimized. A nested PCR approach was used in conjunction with these primer sets to increase the sensitivity of the molecular detection method. Screening of environmental samples indicated that all products amplified by this method were
cld
gene sequences. These sequences were obtained from pristine sites as well as contaminated sites from which DPRB were isolated. More than one
cld
phylotype was also identified from some samples, indicating the presence of more than one DPRB strain at those sites. The use of these primer sets represents a direct and sensitive molecular method for the qualitative detection of (per)chlorate-reducing bacteria in the environment, thus offering another tool for monitoring natural attenuation. Sequences of
cld
genes isolated in the course of this project were also generated from various DPRB and provided the first opportunity for a phylogenetic treatment of this metabolic gene. Comparisons of the
cld
and 16S ribosomal DNA (rDNA) gene trees indicated that the
cld
gene does not track 16S rDNA phylogeny, further implicating the possible role of horizontal transfer in the evolution of (per)chlorate respiration.
Microbial Species Involved in Production of 1,2-
sn
-Diacylglycerol and Effects of Phosphatidylcholine on Human Fecal Microbiota
Authors: Vulevic Jelena , McCartney Anne L. , Gee Jennifer M. , Johnson Ian T. , Gibson Glenn R.
Published: September 2004

1,2-
sn
-Diacylglycerols (DAGs) are activators of protein kinase C (PKC), which is involved in the regulation of colonic mucosal proliferation. Extracellular DAG has been shown to stimulate the growth of cancer cell lines in vitro and may therefore play an important role in tumor promotion. DAG has been detected in human fecal extracts and is thought to be of microbial origin. Hitherto, no attempts have been made to identify the predominant fecal bacterial species involved in its production. We therefore used anaerobic batch culture systems to determine whether fecal bacteria could utilize phosphatidylcholine (0.5% [wt/vol]) to produce DAG. Production was found to be dependent upon the presence of the substrate and was enhanced in the presence of high concentrations of deoxycholate (5 and 10 mM) in the growth medium. Moreover, its production increased with the pH, and large inter- and intraindividual variations were observed between cultures seeded with inocula from different individuals. Clostridia and
Escherichia coli
multiplied in the fermentation systems, indicating their involvement in phosphatidylcholine metabolism. On the other hand, there was a significant decrease in the number of
Bifidobacterium
spp. in the presence of phosphatidylcholine. Pure-culture experiments showed that 10 of the 12 strains yielding the highest DAG levels (>50 nmol/ml) were isolated from batch culture enrichments run at pH 8.5. We found that the strains capable of producing large amounts of DAG were predominantly
Clostridium bifermentans
(8 of 12), followed by
Escherichia coli
(2 of 12). Interestingly, one DAG-producing strain was
Bifidobacterium infantis
, which is often considered a beneficial gut microorganism. Our results have provided further evidence that fecal bacteria can produce DAG and that specific bacterial groups are involved in this process. Future strategies to reduce DAG formation in the gut should target these species.
Increasing the Carbon Flux toward Synthesis of Short-Chain-Length—Medium-Chain-Length Polyhydroxyalkanoate in the Peroxisome of
Saccharomyces cerevisiae
through Modification of the β-Oxidation Cycle
Authors: de Oliveira Valeria Cora , Maeda Isamu , Delessert Syndie , Poirier Yves
Published: September 2004

Short-chain-length-medium-chain-length polyhydroxyalkanoates were synthesized in
Saccharomyces cerevisiae
from intermediates of the β-oxidation cycle by expressing the polyhydroxyalkanoate synthases from
Aeromonas caviae
and
Ralstonia eutropha
in the peroxisomes. The quantity of polymer produced was increased by using a mutant of the β-oxidation-associated multifunctional enzyme with low dehydrogenase activity toward
R
-3-hydroxybutyryl coenzyme A.
Fluorogenic Selective and Differential Medium for Isolation of
Enterobacter sakazakii
Authors: Oh Se-Wook , Kang Dong-Hyun
Published: September 2004

4-Methylumbelliferyl-α-
d
-glucoside, the fluorogenic substrate of α-glucosidase, was used as a selective marker to develop a differential medium for
Enterobacter sakazakii
. This bacterium showed strong fluorogenic characteristics clearly distinguishable from other microorganisms. On the basis of reducing background noise, an optimum basal medium and nitrogen source were selected. Incubation conditions were optimized.
Production of Minicellulosomes from
Clostridium cellulovorans
in
Bacillus subtilis
WB800
Authors: Cho Hee-Yeon , Yukawa Hideaki , Inui Masayuki , Doi Roy H. , Wong Sui-Lam
Published: September 2004

Two genes encoding EngB endoglucanase and mini-CbpA1 scaffolding protein of
Clostridium cellulovorans
were constructed and coexpressed in
Bacillus subtilis
WB800. The resulting minicellulosomes were isolated by gel filtration chromatography and characterized. Biochemical and immunological evidence indicated that fraction II contained minicellulosomes consisting of mini-CbpA1 and EngB. The in vivo synthesis of minicellulosomes suggests that it will be possible in the future to insert into
B. subtilis
cellulosomal genes that will allow growth on cellulosic materials and the production of various designer cellulosomes with specific functions.
Native, not nitrated, cytochrome c and mitochondria-derived hydrogen peroxide drive osteoclast apoptosis
Authors: Oursler Merry Jo , Bradley Elizabeth W. , Elfering Sarah L. , Giulivi Cecilia
Published: September 2004

Two unresolved aspects of the role of mitochondria-derived cytochrome c in apoptosis are whether there is a separate pool of cytochrome c within mitochondria that participates in the activation of apoptosis and whether a chemically modified cytochrome c drives apoptosis. These questions were investigated using osteoclasts, because they are rich in mitochondria and because osteoclast apoptosis is critical in bone metabolism regulation. H2O2 production was increased during culture, preceding cytochrome c release; both processes occurred anterior to apoptosis. With the addition of a mitochondrial uncoupler, H2O2 production and apoptosis were blocked, indicating the prominent role of mitochondria-derived H2O2. Trapping H2O2-derived hydroxyl radical decreased apoptosis. Cytosolic cytochrome c was originated from a single mitochondrial compartment, supporting a common pool involved in respiration and apoptosis, and it was chemically identical to the native form, with no indication of oxidative or nitrative modifications. Protein levels of Bcl-2 and Bc-xL were decreased before apoptosis, whereas expression of wild-type Bcl-2 repressed apoptosis, confirming that cytochrome c release is critical in initiating apoptosis. Cytosolic cytochrome c participated in activating caspase-3 and -9, both required for apoptosis. Collectively, our data indicate that the mitochondria-dependent apoptotic pathway is one of the major routes operating in osteoclasts.
Serum and glucocorticoid-regulated kinase Sgk1 inhibits insulin-dependent activation of phosphomannomutase 2 in transfected COS-7 cells
Authors: Menniti Miranda , Iuliano Rodolfo , Amato Rosario , Boito Rosalia , Corea Monica , Le Pera Ilaria , Gulletta Elio , Fuiano Giorgio , Perrotti Nicola
Published: September 2004

Serum- and glucocorticoid-regulated kinase (Sgk1) is considered to be an essential convergence point for peptide and steroid regulation of ENaC-mediated sodium transport. We tried to identify molecular partners of Sgk1 by yeast two-hybrid screening. Yeast two-hybrid screening showed a specific interaction between Sgk1 and phosphomannomutase (PMM)2, the latter of which is an enzyme involved in the regulation of glycoprotein biosynthesis. The interaction was confirmed in intact cells by coimmunoprecipitation and colocalization detected using confocal microscopy. We were then able to demonstrate that Sgk1 phosphorylated PMM2 in an in vitro assay. In addition, we found that the enzymatic activity of PMM2 is upregulated by insulin treatment and that Sgk1 completely inhibits PMM2 activity both in the absence and in the presence of insulin stimulation. These data provide evidence suggesting that Sgk1 may modulate insulin action on the cotranslational glycosylation of glycoproteins.
Barrier role of actin filaments in regulated mucin secretion from airway goblet cells
Authors: Ehre Camille , Rossi Andrea H. , Abdullah Lubna H. , De Pestel Kathleen , Hill Sandra , Olsen John C. , Davis C. William
Published: September 2004

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/Gq-coupled P2Y2 receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of β- and γ-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of β- or γ-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca2+-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.
Activation of proteinase-activated receptor-1 inhibits neurally evoked chloride secretion in the mouse colon in vitro
Authors: Buresi Michelle C. , Vergnolle Nathalie , Sharkey Keith A. , Keenan Catherine M. , Andrade-Gordon Patricia , Cirino Giuseppe , Cirillo Donatella , Hollenberg Morley D. , MacNaughton Wallace K.
Published: September 2004

The proteinase-activated thrombin receptor-1 (PAR-1) belongs to a unique family of G protein-coupled receptors activated by proteolytic cleavage. We studied the effect of PAR-1 activation in the regulation of ion transport in mouse colon in vitro. Expression of PAR-1 in mouse colon was assessed by RT-PCR and immunohistochemistry. To study the role of PAR-1 activation in chloride secretion, mouse colon was mounted in Ussing chambers. Changes in short-circuit current ( Isc) were measured in tissues exposed to either thrombin, saline, the PAR-1-activating peptide TFLLR-NH2, or the inactive reverse peptide RLLFT-NH2, before electrical field stimulation (EFS). Experiments were repeated in the presence of either a PAR-1 antagonist or in PAR-1-deficient mice to assess receptor specificity. In addition, studies were conducted in the presence of chloride-free buffer or the muscarinic antagonist atropine to assess chloride dependency and the role of cholinergic neurons in the PAR-1-induced effect. PAR-1 mRNA was expressed in full-thickness specimens and mucosal scrapings of mouse colon. PAR-1 immunoreactivity was found on epithelial cells and on neurons in submucosal ganglia where it was colocalized with both VIP and neuropeptide Y. After PAR-1 activation by thrombin or TFLLR-NH2, secretory responses to EFS but not those to forskolin or carbachol were significantly reduced. The reduction in the response to EFS was not observed in the presence of the PAR-1 antagonist, in PAR-1-deficient mice, when chloride was excluded from the bathing medium, or when atropine was present. PAR-1 is expressed in submucosal ganglia in the mouse colon and its activation leads to a decrease in neurally evoked epithelial chloride secretion.
Contrasting effects of ischemia on the kinetics of membrane voltage and intracellular calcium transient underlie electrical alternans
Authors: Lakireddy Vikram , Baweja Paramdeep , Syed Asma , Bub Gil , Boutjdir Mohamed , El-Sherif Nabil
Published: September 2004

Repolarization alternans has been considered a strong marker of electrical instability. The objective of this study was to investigate the hypothesis that ischemia-induced contrasting effects on the kinetics of membrane voltage and intracellular calcium transient (CaiT) can explain the vulnerability of the ischemic heart to repolarization alternans. Ischemia-induced changes in action potential (AP) and CaiT resulting in alternans were investigated in perfused Langendorff guinea pig hearts subjected to 10–15 min of global no-flow ischemia followed by 10–15 min of reperfusion. The heart was stained with 100 μl of rhod-2 AM and 25 μl of RH-237, and AP and CaiT were simultaneously recorded with an optical mapping system of two 16 × 16 photodiode arrays. Ischemia was associated with shortening of AP duration (D) but delayed upstroke, broadening of peak, and slowed decay of CaiT resulting in a significant increase of CaiT-D. The changes in APD were spatially heterogeneous in contrast to a more spatially homogeneous lengthening of CaiT-D. CaiT alternans could be consistently induced with the introduction of a shorter cycle when the upstroke of the AP occurred before complete relaxation of the previous CaiT and generated a reduced CaiT. However, alternans of CaiT was not necessarily associated with alternans of APD, and this was correlated with the degree of spatially heterogeneous shortening of APD. Sites with less shortening of APD developed alternans of both CaiT and APD, whereas sites with greater shortening of APD could develop a similar degree of CaiT alternans but slight or no APD alternans. This resulted in significant spatial dispersion of APD. The study shows that the contrasting effects of ischemia on the duration of AP and CaiT and, in particular, on their spatial distribution explain the vulnerability of ischemic heart to alternans and the increased dispersion of repolarization during alternans.
Morphine mimics the antiapoptotic effect of preconditioning via an Ins(1,4,5)P3 signaling pathway in rat ventricular myocytes
Authors: Barrère-Lemaire Stéphanie , Combes Nicolas , Sportouch-Dukhan Catherine , Richard Sylvain , Nargeot Joël , Piot Christophe
Published: September 2004

Morphine has cardioprotective effects against ischemic-reperfusion injuries. This study investigates whether morphine could mimic the antiapoptotic effect of preconditioning using a model of cultured neonatal rat cardiomyocytes subjected to metabolic inhibition (MI). To quantify MI-induced apoptosis, DNA fragmentation and mitochondrial cytochrome c release levels were measured by ELISA. MI-dependent DNA fragmentation was prevented by both Z-VAD-fmk (20 μM), a pan-caspase inhibitor, and cyclosporine A (CsA; 5 μM), a mitochondrial pore transition blocker, added during MI (36% and 54% decrease, respectively). MI-dependent cytochrome c release was not blocked by Z-VAD-fmk but was decreased (38%) by CsA during MI. Metabolic preconditioning (MIP) and preconditioning with morphine (1 μM) were also assessed. MI-dependent DNA fragmentation and cytochrome c release were prevented by MIP (40% and 45% decrease, respectively) and morphine (34% and 45%, respectively). The antiapoptotic effect of morphine was abolished by naloxone (10 nM), a nonselective opioid receptor antagonist, or xestospongin C (XeC, 400 nM), an inhibitor of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]-mediated Ca2+ release. Ca2+ preconditioning, induced by increasing extracellular Ca2+ from 1.8 to 3.3 mM, mimicked the antiapoptotic effect of morphine on DNA fragmentation (24% decrease) and cytochrome c release (57% decrease). This effect mediated by extracellular Ca2+ was also abolished by XeC. Measurements of intracellular Ca2+ concentration using fura-2 microspectrofluorimetry showed that morphine induces Ins(1,4,5)P3-dependent Ca2+ transients abolished by 2-aminoethoxydiphenyl borate (2-APB), a cell-permeable Ins(1,4,5)P3 antagonist. These results suggest that morphine preconditioning prevents simulated ischemia-reperfusion-induced apoptosis via an Ins(1,4,5)P3 signaling pathway in rat ventricular myocytes.
Ammonia production and secretion by S3 proximal tubule segments from acidotic mice: role of ANG II
Authors: Nagami Glenn T.
Published: September 2004

ANG II has potent effects on ammonia production and secretion rates by the proximal tubule and is found in substantial concentrations in the lumen of the proximal tubule in vivo. Because our previous studies demonstrated that acid loading enhanced the stimulatory effects of ANG II on ammonia production and secretion by S2 proximal tubule segments, we examined the effect of ANG II on ammonia production and secretion by isolated, perfused S3 segments from nonacidotic control mice and acidotic mice given NH4Cl for 7 days. In the absence of ANG II, ammonia production and secretion rates were no different in S3 segments from acidotic and control mice. By contrast, when ANG II was present in the luminal perfusion solution, ammonia production and secretion rates were stimulated, in a losartan-inhibitable manner, to a greater extent in S3 segments from acidotic mice. Ammonia secretion rates in S3 segments were largely inhibited by perfusion with a low-sodium solution containing amiloride in the presence or absence of ANG II. These results demonstrated that isolated, perfused mouse S3 proximal tubule segments produce and secrete ammonia, that NH4Cl-induced acidosis does not affect the basal rates of ammonia production and secretion, and that ANG II, added to the luminal fluid, stimulates ammonia production and secretion to a greater extent in S3 segments from acidotic mice. These findings suggest that S3 segments, in the presence of ANG II, can contribute to the enhanced renal excretion that occurs with acid loading.
Sodium and potassium handling by the aldosterone-sensitive distal nephron: the pivotal role of the distal and connecting tubule
Authors: Meneton Pierre , Loffing Johannes , Warnock David G.
Published: September 2004

Sodium reabsorption and potassium secretion in the distal convoluted tubule and in the connecting tubule can maintain the homeostasis of the body, especially when dietary sodium intake is high and potassium intake is low. Under these conditions, a large proportion of the aldosterone-regulated sodium and potassium transport would occur in these nephron segments before the tubular fluid reaches the collecting duct. The differences between these two segments and the collecting duct would be more quantitative than qualitative. The collecting duct would come into play when the upstream segments are overloaded by a primary genetic defect that affects sodium and/or potassium transport or by a diet that is exceedingly poor in sodium and rich in potassium. It is likely that the homeostatic role of the distal convoluted and connecting tubules, which are technically difficult to study, has been underestimated, whereas the role of the more easily accessible collecting duct may have been overemphasized.
A Comparison of Neuron Response Properties in Areas A1 and CM of the Marmoset Monkey Auditory Cortex: Tones and Broadband Noise
Authors: Kajikawa Yoshinao , de La Mothe Lisa , Blumell Suzanne , Hackett Troy A.
Published: September 2004

The purpose of this study was to compare response properties of two adjacent areas of the marmoset monkey auditory cortex. Multiunit responses to 50 ms tones and broadband noise bursts (BBN) were recorded in the core area, A1, and the caudomedial belt area, CM, of ketamine-anesthetized animals. Neurons in A1 and CM exhibited robust low-threshold short-latency responses to BBN and tones, whereas neurons in adjoining lateral belt areas were poorly responsive or unresponsive to tones and noise. Except for a population of broadly tuned units in CM, the characteristic frequency (CF) could be determined for all recording sites in A1 and CM. Both areas were tonotopically organized and shared a high CF border. Whereas the tonotopic gradient in A1 was smooth and continuous across the field, the gradient in CM was discontinuous, and the intermediate CF range was underrepresented. For BBN stimuli, rate level functions were largely monotonic in A1 and CM. Response profiles were also similar in both areas. As a population, neurons in CM were distinguished from A1 by significantly shorter response latencies, lower thresholds, and broader tuning bandwidth at higher intensities. The results indicated that, while A1 and CM represent anatomically and physiologically distinct areas, their response profiles under anesthesia overlapped considerably compared with the lateral belt areas. Therefore refinements of current models of the primate auditory cortex may be needed to account for differences in organization among the auditory belt areas.
The Speed of Auditory Low-Side Suppression
Authors: van der Heijden Marcel , Joris Philip X.
Published: September 2004

The nonlinear cochlear phenomenon of two-tone suppression is known to be very fast, but precisely how fast is unknown. We studied the timing of low-side suppression in the auditory nerve of the cat using multitone complexes as auditory stimuli. An evalution of the group delays of the responses to these complexes allowed us to measure the timing of the responses with sub-millisecond accuracy for a large number of fibers with characteristic frequencies (CFs) between 2 and 40 kHz. In particular, we measured the delays with which the same below-CF tone complexes affected the response either as an excitor (when presented alone) or as a suppressor (when combined with a CF probe). For CFs <10 kHz, we found that the delay of suppression was larger than the delay of excitation by several hundred microseconds. The difference between the delay of suppression and that of excitation decreased with increasing CF, becoming negligible for CFs >15 kHz. The results are analyzed in terms of traveling-wave delays and a purported cochlear gain control. The data suggest that suppression originates from a gain-control mechanism with an integration time in the order of two cycles of CF.
Tone Frequency Maps and Receptive Fields in the Developing Chinchilla Auditory Cortex
Authors: Pienkowski Martin , Harrison Robert V.
Published: September 2004

Single-unit responses to tone pip stimuli were isolated from numerous microelectrode penetrations of auditory cortex (under ketamine anesthesia) in the developing chinchilla ( laniger), a precocious mammal. Results are reported at postnatal day 3 (P3), P15, and P30, and from adult animals. Hearing sensitivity and spike firing rates were mature in the youngest group. The topographic representation of sound frequency (tonotopic map) in primary and secondary auditory cortex was also well ordered and sharply tuned by P3. The spectral-temporal complexity of cortical receptive fields, on the other hand, increased progressively (past P30) to adulthood. The (purported) refinement of initially diffuse tonotopic projections to cortex thus seems to occur in utero in the chinchilla, where external (and maternal) sounds are considerably attenuated and might not contribute to the mechanism(s) involved. This compares well with recent studies of vision, suggesting that the refinement of the retinotopic map does not require external light, but rather waves of (correlated) spontaneous activity on the retina. In contrast, it is most probable that selectivity for more complex sound features, such as frequency stacks and glides, develops under the influence of the postnatal acoustic environment and that inadequate sound stimulation in early development (e.g., due to chronic middle ear disease) impairs the formation of the requisite intracortical (and/or subcortical) circuitry.
Role of Mossy Fiber Sprouting and Mossy Cell Loss in Hyperexcitability: A Network Model of the Dentate Gyrus Incorporating Cell Types and Axonal Topography
Authors: Santhakumar Vijayalakshmi , Aradi Ildiko , Soltesz Ivan
Published: September 2004

Mossy cell loss and mossy fiber sprouting are two characteristic consequences of repeated seizures and head trauma. However, their precise contributions to the hyperexcitable state are not well understood. Because it is difficult, and frequently impossible, to independently examine using experimental techniques whether it is the loss of mossy cells or the sprouting of mossy fibers that leads to dentate hyperexcitability, we built a biophysically realistic and anatomically representative computational model of the dentate gyrus to examine this question. The 527-cell model, containing granule, mossy, basket, and hilar cells with axonal projections to the perforant-path termination zone, showed that even weak mossy fiber sprouting (10–15% of the strong sprouting observed in the pilocarpine model of epilepsy) resulted in the spread of seizure-like activity to the adjacent model hippocampal laminae after focal stimulation of the perforant path. The simulations also indicated that the spatially restricted, lamellar distribution of the sprouted mossy fiber contacts reported in in vivo studies was an important factor in sustaining seizure-like activity in the network. In contrast to the robust hyperexcitability-inducing effects of mossy fiber sprouting, removal of mossy cells resulted in decreased granule cell responses to perforant-path activation in agreement with recent experimental data. These results indicate the crucial role of mossy fiber sprouting even in situations where there is only relatively weak mossy fiber sprouting as is the case after moderate concussive experimental head injury.
Rapid Identification of Dysregulated Genes in Cutaneous Malignant Melanoma Metastases Using cDNA Technology
Authors: Mirmohammadsadegh Alireza , Baer Annett , Nambiar Sandeep , Bardenheuer Walter , Hengge Ulrich R.
Published: September 2004

One important application of DNA microarray technology is the simultaneous analysis of gene expression of different mRNAs. Comparison of mRNA patterns of diseased and healthy tissue may help to understand the pathogenesis of a given disorder. In cancer tissue, identified dysregulated genes may serve as new molecular markers for diagnosis or prognosis or may ideally serve as new targets for therapy. Using membrane cDNA array technology, we analyzed gene expression in human melanomas, one of the most aggressive types of cancer with a high metastatic potential and with markedly increased incidence worldwide. To account for the heterogeneity of tumors, we compared total RNA from cutaneous melanoma metastases of 10 different patients with primary human melanocytes. An abundance of genes was dysregulated (up-/downregulated), which involved for example the apoptosis gene growth factor receptor-bound protein 10, Bcl2-associated X membrane protein, Bcl2 antagonist of cell death, glutathione S-transferase θ1 and glutathione reductase. Ultimately, the identification of melanoma-associated genes may provide a potential therapeutic strategy for identifying and targeting malignant melanoma.
Exogenous siRNA Mediates Sequence-Independent Gene Suppression by Signaling through Toll-Like Receptor 3
Authors: Karikó Katalin , Bhuyan Prakash , Capodici John , Ni Houping , Lubinski John , Friedman Harvey , Weissman Drew
Published: September 2004

RNA interference (RNAi) is a powerful method that specifically suppresses gene expression in a sequence-dependent manner whose machinery is found in organisms from fungi to mammals. Mammalian cells have developed a sequence-independent system of gene suppression often induced by viral replication that includes the recognition of double-stranded RNA (dsRNA) through Toll-like receptor 3 (TLR3) and induction of type I interferon synthesis. Interferon activates the transcription of a set of genes including dsRNA-activated protein kinase that suppresses protein synthesis and 2′-5′-oligoadenylate synthetase, which generates a product that activates RNase L to cleave RNA in a sequence-independent manner. We observed that 21-bp dsRNA, a key mediator of RNAi, not only induces sequence-specific gene suppression, but also signals TLR3 to induce type I interferon and activates sequence-independent suppression of protein synthesis and enhancement of mRNA degradation. This sequence-independent suppression was demonstrated for both an exogenously administered reporter gene as well as during the targeting of viral genes in the course of acute herpes simplex virus type I infection of keratinocytes. As TLR3 is expressed by many primary cell types and cell lines, this sequence-independent suppression should be considered in the design of experiments using small interfering RNA-mediated gene suppression.
Experimental Leishmania major Infection Suppresses HIV-1 DNA Vaccine Induced Cellular Immune Response
Authors: Robinson Tara M. , Nelson Robin , Artis David , Scott Phillip , Boyer Jean D.
Published: September 2004

The AIDS epidemic in the developing world represents a major global crisis and an effective vaccine is imperative. However, many parasites are common in developing countries and can result in a state of chronic immune activation that is polarized towards a Th2 profile and which can potentially impair responses to vaccines or other infectious challenges. In this study we demonstrate that experimental Leishmania major infection of BALB/c mice inhibits responses to a DNA-based HIV-1 gag vaccine. L. major infection in BALB/c results in a polarized Th2 immune response. In this study naïve BALB/c mice immunized with the HIV-1 gag DNA vaccine mounted a cellular immune response against the vaccine antigen, HIV-1 gag. CD8+ T lymphocytes were able to respond in vitro to HIV-1 gag stimulation and secrete interferon (IFN)-γ. However, L. major-infected, vaccinated BALB/c mice had a significantly reduced number of IFN-γ-producing CD8+ T cells following in vitro stimulation with gag antigen. These data suggest that parasitic infection, which results in a Th2 profile, reduces the efficacy of DNA vaccines that are designed to induce antiviral CD8+ T cell responses.
Family History of Coronary Artery Disease and Prognosis after First Acute Myocardial Infarction in a National Survey
Authors: Harpaz David , Behar Solomon , Rozenman Yoseph , Boyko Valentina , Gottlieb Shmuel
Published: September 2004

A positive family history (FH) of coronary artery disease (CAD) is considered an independent risk factor for developing CAD. However, the natural history, coronary angiographic findings and prognosis of patients with a positive FH developing first acute myocardial infarction (AMI) are not well defined. A cohort of 2,690 consecutive patients with first AMI from two prospective nationwide surveys conducted during 1996 and 1998 in all coronary care units operating in Israel was studied. Baseline characteristics, hospital course, management and outcome of 405 patients with first AMI and a positive FH were compared with 2,285 controls without a positive FH. Coronary angiograms of patients with and without a positive FH were reviewed and compared. Patients with a positive FH were younger (53 vs. 64 years), more often male, current smokers and patients with hyperlipidemia, but less often patients with diabetes or hypertension than patients without a positive FH. Patients with a positive FH developed heart failure during hospital stay less frequently. Thrombolytic therapy was similarly administered to both groups. During the hospital stay, coronary angiography, percutaneous coronary intervention or coronary artery bypass grafting were more frequently performed in patients with a positive FH. The coronary anatomy and the extent of the CAD were similar in patients with and without a positive FH. Crude and covariate-adjusted mortality rates were significantly lower in patients with a positive FH than in patients without a positive FH on day 30 (2.2 vs. 9.6%, p < 0.001; odds ratio 0.50, 95% confidence interval 0.22–0.99) and at 1 year (3.5 vs. 14%, p < 0.001; hazard ratio 0.58, 95% confidence interval 0.42–0.80). Patients with a positive FH developed their first AMI more than 1 decade earlier in comparison to those without such a history. The extent of their coronary disease is similar to the older patients without a positive FH. The better prognosis of patients with a positive FH is mostly explained by their younger age.
Composite Valve Graft versus Separate Aortic Valve and Ascending Aortic Replacement
Authors: Brandt Michael , Abdelkerim Someia , Clemm Susanne , Böning Andreas , Cremer Jochen
Published: September 2004

To ascertain if the operative technique has any influence on outcome, the surgical results after aortic root replacement using either a composite valve graft (CVG) or a separate graft and valve (SVG) were analyzed. Eighty-four patients received a CVG, and 36 had SVG replacement. The operative mortality rate was 6% for patients receiving a CVG and 3% for SVG replacement (nonsignificant). Follow-up extended to 21 years (mean 124 ± 45 months). The type of the procedure (SVG versus CVG) was not a significant predictor of in-hospital mortality, length of hospital stay, subsequent root dilatation (SVG), anastomotic dehiscence and subsequent surgery. The early and long-term results after CVG or SVG were similar, which reflects proper patient selection.
Role of cAMP Signaling in the Mediation of Dopamine-Induced Stimulation of GnRH Secretion via D1 Dopamine Receptors in GT1-7 Cells
Authors: Yoshida Hiroshi , Paruthiyil Sreenivasan , Butler Paul , Weiner Richard I.
Published: September 2004

Pharmacologically increasing cyclic adenosine monophosphate (cAMP) levels in GT1 gonadotropin-releasing hormone (GnRH) cell lines increased the secretion of GnRH. Dopamine (DA) increased the GnRH secretion in GT1 cells via a DA receptor positively coupled to adenylate cyclase. We then asked whether inhibition of the DA-induced increase in cAMP would block the stimulatory effect of DA on GnRH release. Expression of the cAMP-specific phosphodiesterase (PDE4D1) was used in a genetic approach to inhibit the DA-induced increase in cAMP levels. Cells were infected with an adenovirus vector (Ad) expressing PDE4D1 (PDE-Ad) or, for controls, with an empty Ad (Null-Ad). Infection with the PDE-Ad completely blocked the forskolin-induced stimulation of GnRH secretion and [Ca2+]i and decreased the majority of the release of cAMP into the culture medium. In contrast, although PDE-Ad infection blocked virtually all of the DA-induced increase in extracellular cAMP, the release of GnRH and the increase in [Ca2+]i were only delayed for approximately 15 min. GT1 cells express the D1 DA receptor which is positively coupled to adenylate cyclase but not the D5 DA receptor. These data suggest that the initial phase of the DA-induced secretion of GnRH is dependent on an increase in cAMP levels. However, it appears that an additional non-cAMP-regulated signaling pathway is involved in the stimulation of GnRH release via the D1 DA receptor.
T Regulatory Cells in Allergy and Health: A Question of Allergen Specificity and Balance
Authors: Taylor Alison , Verhagen Johan , Akdis Cezmi A. , Akdis Mübeccel
Published: September 2004

Anergy, tolerance and active suppression may not be independent events, but rather involve similar mechanisms and cell types in immune regulation. Induction of allergen-specific regulatory/suppressor T cells (TReg) seems essential for the maintenance of a healthy immune response to allergens. Allergen-specific immunotherapy can induce specific TReg cells that abolish allergen-induced proliferation of T helper 1 (Th1) and Th2 cells, as well as their cytokine production. TReg cells utilize multiple suppressive mechanisms, interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) as secreted cytokines and CTLA-4, PD-1, mTGF-β, mIL-10, TGF-βR and IL-10R as surface molecules. An important aspect of TReg cells is the regulation of antibody isotypes and suppression of proinflammatory cells. IL-10 and TGF-β secreted by TReg cells skew production of IgE towards the noninflammatory isotypes, IgG4 and IgA, respectively. Furthermore, TReg cells may directly or indirectly suppress effector cells of allergic inflammation such as basophils and eosinophils. In conclusion, induction of antigen-specific TReg cells may redirect an inappropriate immune response against allergen or autoantigens with the help of a broad range of suppressor mechanisms.
Effect of 12-Month Therapy with Omega–3 Polyunsaturated Acids on Glomerular Filtration Response to Dopamine in IgA Nephropathy
Authors: Sulikowska Beata , Niewęgłowski Tomasz , Manitius Jacek , Łysiak-Szydłowska Wiesława , Rutkowski Bolesław
Published: September 2004

Background: ω–3 polyunsaturated acids therapy is efficient in primary IgA nephropathy. It is unknown whether doses of ω–3 smaller than those given previously are still effective. The aim of the study was to examine the effect of ω–3 therapy on renal vascular function in relation to proteinuria and urinary excretion of N-acetyl-β-D-glucosaminidase (NAG). Methods: 20 IgA patients aged 36.5 ± 10.77 with creatinine clearance (Crcl) 105.71 ± 27.3 ml/min and proteinuria 3.31 ± 2.01 g/24 h were given orally 810 mg EPA and 540 mg DHA daily for 12 months. Before and at the end of the study, 24-hour proteinuria, serum homocysteine, and Crcl were measured. At the same time, renal vascular function was estimated as dopamine-induced glomerular filtration response (DIR). DIR was measured as: two 120-min lasting Crcl (before and during 2 µg/kg b.w./min i.v. dopamine). Results: The results obtained during follow-up were as follows (baseline vs. after therapy): DIR 14.9 ± 16.4 vs. 30.3 ± 14.3% (p < 0.01); urine protein 2.31 ± 2.01 vs. 1.31 ± 1.37 g/24 h (p < 0.01); (Crcl) 105.71 ± 27.3 vs. 103.9 ± 20.9 ml/min (n.s.); NAG 8.3 ± 1.8 vs. 6.0 ± 1.2 U/gcreat (p < 0.01), and homocysteine 16.2 ± 3.15 vs. 13.8 ± 2.6 µmol/l (p < 0.05). The only correlation found was linear correlation between basal DIR and DIR change (r = –0.570; p < 0.010) and basal NAG (r = –0.460; p < 0.50). Conclusions: ω–3 supplementation is associated with the improvement of both renal vascular function and tubule function.
The Fruit of Urban Nature
Authors: Sullivan William C. , Kuo Frances E. , Depooter Stephen F.
Published: September 2004

What makes a neighborhood space vital? This article explores the possibility that the presence of trees and grass may be one of the key components of vital neighborhood spaces. We report on 758 observations of individuals in 59 outdoor common spaces in a residential development. Twenty-seven of the neighborhood common spaces were relatively green, whereas 32 were relatively barren. Results indicate that the presence of trees and grass is related to the use of outdoor spaces, the amount of social activity that takes place within them, and the proportion of social to nonsocial activities they support. The findings improve and broaden our understanding of the physical characteristics that influence social contact among neighbors and provide evidence that nature plays an important role in creating vital neighborhood spaces.