bluuebunny/crossref_metadata_2025 huggingface dataset:
Microtubule Motor Ncd Induces Sliding of Microtubules In Vivo
Authors: Oladipo Abiola , Cowan Ann , Rodionov Vladimir
Published: June 2007

The mitotic spindle is a microtubule (MT)-based molecular machine that serves for equal segregation of chromosomes during cell division. The formation of the mitotic spindle requires the activity of MT motors, including members of the kinesin-14 family. Although evidence suggests that kinesins-14 act by driving the sliding of MT bundles in different areas of the spindle, such sliding activity had never been demonstrated directly. To test the hypothesis that kinesins-14 can induce MT sliding in living cells, we developed an in vivo assay, which involves overexpression of the kinesin-14 family member Drosophila Ncd in interphase mammalian fibroblasts. We found that green fluorescent protein (GFP)–Ncd colocalized with cytoplasmic MTs, whose distribution was determined by microinjection of Cy3 tubulin into GFP-transfected cells. Ncd overexpression resulted in the formation of MT bundles that exhibited dynamic “looping” behavior never observed in control cells. Photobleaching studies and fluorescence speckle microscopy analysis demonstrated that neighboring MTs in bundles could slide against each other with velocities of 0.1 μm/s, corresponding to the velocities of movement of the recombinant Ncd in in vitro motility assays. Our data, for the first time, demonstrate generation of sliding forces between adjacent MTs by Ncd, and they confirm the proposed roles of kinesins-14 in the mitotic spindle morphogenesis.
Syk-dependent Actin Dynamics Regulate Endocytic Trafficking and Processing of Antigens Internalized through the B-Cell Receptor
Authors: Le Roux Delphine , Lankar Danielle , Yuseff Maria-Isabel , Vascotto Fulvia , Yokozeki Takeaki , Faure-André Gabrielle , Mougneau Evelyne , Glaichenhaus Nicolas , Manoury Bénédicte , Bonnerot Christian , Lennon-Duménil Ana-Maria
Published: June 2007

Antigen binding to the B-cell receptor (BCR) induces multiple signaling cascades that ultimately lead to B lymphocyte activation. In addition, the BCR regulates the key trafficking events that allow the antigen to reach endocytic compartments devoted to antigen processing, i.e., that are enriched for major histocompatibility factor class II (MHC II) and accessory molecules such as H2-DM. Here, we analyze the role in antigen processing and presentation of the tyrosine kinase Syk, which is activated upon BCR engagement. We show that convergence of MHC II- and H2-DM–containing compartments with the vesicles that transport BCR-uptaken antigens is impaired in cells lacking Syk activity. This defect in endocytic trafficking compromises the ability of Syk-deficient cells to form MHC II-peptide complexes from BCR-internalized antigens. Altered endocytic trafficking is associated to a failure of Syk-deficient cells to properly reorganize their actin cytoskeleton in response to BCR engagement. We propose that, by modulating the actin dynamics induced upon BCR stimulation, Syk regulates the positioning and transport of the vesicles that carry the molecules required for antigen processing and presentation.
Dynamic Regulation of the Large Exocytotic Fusion Pore in Pancreatic Acinar Cells
Authors: Larina Olga , Bhat Purnima , Pickett James A. , Launikonis Bradley S. , Shah Amit , Kruger Wade A. , Edwardson J. Michael , Thorn Peter
Published: June 2007

Loss of granule content during exocytosis requires the opening of a fusion pore between the secretory granule and plasma membrane. In a variety of secretory cells, this fusion pore has now been shown to subsequently close. However, it is still unclear how pore closure is physiologically regulated and contentious as to how closure relates to granule content loss. Here, we examine the behavior of the fusion pore during zymogen granule exocytosis in pancreatic acinar cells. By using entry of high-molecular-weight dyes from the extracellular solution into the granule lumen, we show that the fusion pore has a diameter of 29–55 nm. We further show that by 5 min after granule fusion, many granules have a closed fusion pore with evidence indicating that pore closure is a prelude to endocytosis and that in granules with a closed fusion pore the chymotrypsinogen content is low. Finally, we show that latrunculin B treatment promotes pore closure, suggesting F-actin affects pore dynamics. Together, our data do not support the classical view in acinar cells that exocytosis ends with granule collapse. Instead, for many granules the fusion pore closes, probably as a transition to endocytosis, and likely involving an F-actin–dependent mechanism.
Rho/Rho-associated Kinase-II Signaling Mediates Disassembly of Epithelial Apical Junctions
Authors: Samarin Stanislav N. , Ivanov Andrei I. , Flatau Gilles , Parkos Charles A. , Nusrat Asma
Published: June 2007

Apical junctional complex (AJC) plays a vital role in regulation of epithelial barrier function. Disassembly of the AJC is observed in diverse physiological and pathological states; however, mechanisms governing this process are not well understood. We previously reported that the AJC disassembly is driven by the formation of apical contractile acto-myosin rings. In the present study, we analyzed the signaling pathways regulating acto-myosin–dependent disruption of AJC by using a model of extracellular calcium depletion. Pharmacological inhibition analysis revealed a critical role of Rho-associated kinase (ROCK) in AJC disassembly in calcium-depleted epithelial cells. Furthermore, small interfering RNA (siRNA)-mediated knockdown of ROCK-II, but not ROCK-I, attenuated the disruption of the AJC. Interestingly, AJC disassembly was not dependent on myosin light chain kinase and myosin phosphatase. Calcium depletion resulted in activation of Rho GTPase and transient colocalization of Rho with internalized AJC proteins. Pharmacological inhibition of Rho prevented AJC disassembly. Additionally, Rho guanine nucleotide exchange factor (GEF)-H1 translocated to contractile F-actin rings after calcium depletion, and siRNA-mediated depletion of GEF-H1 inhibited AJC disassembly. Thus, our findings demonstrate a central role of the GEF-H1/Rho/ROCK-II signaling pathway in the disassembly of AJC in epithelial cells.
Cell-bionics: tools for real-time sensor processing
Authors: Toumazou Chris , Cass Tony
Published: June 2007

The accurate monitoring of the physiological status of cells, tissues and whole organisms demands a new generation of devices capable of providing accurate data in real time with minimal perturbation of the system being measured. To deliver on the promise ofcell-bionicsadvances over the past decade in miniaturization, analogue signal processing, low-power electronics, materials science and protein engineering need to be brought together. In this paper we summarize recent advances in our research that is moving us in this direction. Two areas in particular are highlighted: the exploitation of the physical properties inherent in semiconductor devices to perform very low power on chip signal processing and the use of gene technology to tailor proteins for sensor applications. In the context of engineered tissues, cell-bionics could offer the ability to monitor the precise physiological state of the construct, both during ‘manufacture’ and post-implantation. Monitoring during manufacture, particularly by embedded devices, would offer quality assurance of the materials components and the fabrication process. Post-implantation monitoring would reveal changes in the underlying physiology as a result of the tissue construct adapting to its new environment.
Root and canal morphology of maxillary first and second permanent molar teeth in a Ugandan population
Authors: Rwenyonyi C. M. , Kutesa A. M. , Muwazi L. M. , Buwembo W.
Published: June 2007

Aim  To investigate the root and canal morphology of permanent maxillary molar teeth from a Ugandan population.

Methodology  Maxillary first (n = 221) and second molar (n = 221) teeth were collected from patients attending dental clinics in Kampala. Teeth were prepared using a clearing technique: the pulp chambers were accessed and the teeth placed consecutively into 5% sodium hypochlorite, 10% nitric acid, then methyl salicylate. Indian ink was injected into the pulp chambers to demonstrate the canal system.

Results  In the first molars, 95.9% of the teeth had separate roots. The mesiobuccal root was fused with the palatal root in 3% of specimens and with the distobuccal root in 0.5% of teeth. In the second molars, 86% of the teeth had separate roots. The mesiobuccal root was fused with the palatal root in 6.3% of specimens and with the distobuccal root in 6.8% of teeth. Apical deltas were more frequent in the mesiobuccal root when compared with distobuccal and palatal roots of both the first and second molars. A type I canal configuration (>75%) was the most frequent in all the roots of both the first and second molars. Canal intercommunications and lateral canals were more frequent in the mesiobuccal root when compared with other roots.

Conclusions  The mesiobuccal root tended to have more variations in the canal system followed by the distobuccal root, whereas the palatal root had the least. The findings in root and canal morphology of this Ugandan population were different from previous studies, which may partly be attributed to racial differences.
Root canal treatment on a patient with zinc oxide allergy: a case report
Authors: Karabucak B. , Stoopler E. T.
Published: June 2007

Aim To describe successful root canal treatment of a patient with a true zinc oxide allergy and to discuss allergic reactions to dental materials.

Summary Dental materials have been reported as aetiologic agents for both local and systemic allergic reactions. It is essential for the oral healthcare provider to recognize the clinical symptoms associated with allergic reactions and to modify dental treatment, if necessary, to prevent these reactions from occurring. This article describes an unusual case of a patient with an allergy to zinc oxide. To our knowledge, this is the first case of successful root canal treatment of a patient with confirmed zinc oxide allergy to be reported in the dental literature.

Key learning points 

Medical and dental histories must be evaluated to prevent medical complications secondary to dental treatment.

Any patient suspected of having an allergy to dental materials should be referred to a healthcare professional capable of performing and interpreting allergy tests prior to dental treatment.
One of two gbpC gene homologues is involved in dextran‐dependent aggregation of Streptococcus sobrinus
Authors: Kagami A. , Okamoto‐Shibayama K. , Yamamoto Y. , Sato Y. , Kizaki H.
Published: June 2007

Introduction:  Streptococcus sobrinus exhibits marked dextran‐dependent aggregation mediated by glucan‐binding proteins (GBPs). In contrast to Streptococcus mutans, in which the gbpC gene responsible for dextran‐dependent aggregation of this organism has been characterized, genes encoding the S. sobrinus GBPs have not yet been identified.

Methods:  Recently, we identified the gbpC gene homologue from Streptococcus macacae using polymerase chain reaction primers based on the conserved regions of the gbpC sequence exhibiting intraspecies variations. This method was applied to amplify a S. sobrinus homologue.

Results:  Unexpectedly, two gbpC gene homologues were identified in S. sobrinus strain 100‐4. One homologue, named gbpC, was more similar to the S. mutans gbpC gene than the other and was approximately half the molecular size of its homologue with similar regions interrupted by several non‐similar stretches. However, the dextran‐binding activity of the protein expressed from gbpC in Escherichia coli was not detected in contrast to the other homologue, a protein designated as Dbl, expressing this activity. The gbpC gene was shown to be intact on the chromosome of strain OMZ176, which does not exhibit dextran‐dependent aggregation, while the dbl gene of this strain contained a single adenine nucleotide insertion at approximately one‐third the distance from the 5′‐end. The insertion mutation in the dbl gene resulted in translation of a premature protein missing its LPXTG sequence signature sequence of the wall‐anchored proteins.

Conclusion:  These results suggest that the dbl gene is very likely responsible for S. sobrinus dextran‐dependent aggregation.
Recovery of Enterococcus faecalis from cheese in the oral cavity of healthy subjects
Authors: Razavi A. , Gmür R. , Imfeld T. , Zehnder M.
Published: June 2007

Introduction:  Enterococci are rarely found in the healthy human oral cavity, yet they are strongly associated with filled root canals. The origin of these enterococci remains unknown. Our hypothesis is that they are transient food‐born colonizers under healthy conditions. This pilot study reinvestigated the prevalence of enterococci in the oral cavity of healthy volunteers, screened cheese samples for enterococci and investigated colonization of the oral cavity after ingestion of an enterocci‐positive cheese.

Method:  Concentrated oral rinse samples were collected from a cohort of 50 dental students and proved negative for viable enterococci. Twenty cheese samples were obtained from local supermarkets. Enterococci were cultured and identified using standard methods.

Results:  Viable enterococci were detected in one of five specimens of Swiss Tilsiter, three of five samples of French soft cheese, one of five Mozzarella samples and one of five Feta samples. Eight volunteers from the cohort consumed 10 g of a cheese with high Enterococcus faecalis load. Oral rinse samples were collected before and 1, 10 and 100 min after cheese ingestion. One minute after ingestion, a median of 5480 E. faecalis colony‐forming units was recovered from the oral rinse samples. Bacterial counts were reduced after 10 min, had dropped after 100 min to levels that were significantly (P < 0.005) different from the 1‐min and 10‐min scores and were below the detection limit after 1 week.

Conclusions:  These findings suggest that colonization of the healthy oral cavity by enterococci is transitional, but at the same time add weight to our hypothesis that enterococcal root canal infections could be food‐borne.
Cultivable bacteria in infected root canals as identified by 16S rRNA gene sequencing
Authors: Siqueira J. F. , Rôças I. N. , Paiva S. S. M. , Magalhães K. M. , Guimarães‐Pinto T.
Published: June 2007

Introduction:  Traditionally, cultivable bacteria isolated from infected root canals have been identified by phenotype‐based methods. Because 16S ribosomal RNA (rRNA) gene sequencing has emerged as a more accurate and reliable tool for bacterial identification, the present study applied this approach to identify bacterial isolates recovered from the root canals of teeth with chronic apical periodontitis.

Methods:  Anaerobic techniques were used for culturing; identification of the isolates was carried out by polymerase chain reaction amplification and sequencing of the V5–V8 region of the 16S rRNA gene. Bacteria were found in all samples. The mean number of taxa per canal was 3.1, ranging from 2 to 8. The median number of cultivable bacterial cells in the root canals was 4.2 × 105, ranging from 2.8 × 103 to 3.3 × 107. Eighty‐seven strains belonging to 52 bacterial taxa were identified. The most prevalent taxa were Fusobacterium nucleatum, Porphyromonas gingivalis, Pseudoramibacter alactolyticus, Micromonas micros and streptococci. The following bacterial phyla were represented in this study: Firmicutes (22 taxa, 46% of the identified isolates), Actinobacteria (14 taxa, 25.3% of the isolates), Bacteroidetes (eight taxa, 13.8% of the isolates), Fusobacteria (three taxa, 9.2% of the isolates) and Proteobacteria (five taxa, 5.7% of the isolates). Some of the isolates represented unnamed species not previously cultivated and characterized. In conclusion, our findings using a combined anaerobic culture–molecular identification approach confirmed the polymicrobial nature of primary endodontic infections with dominance of anaerobic bacteria. Notably several bacteria that are difficult or impossible to identify by phenotypic means were identified, including previously uncultivated taxa, cultivated‐but‐not‐yet‐characterized taxa and newly named species.
IL‐1β inhibits IK and increases [Ca2+]i in the carotid body glomus cells and increases carotid sinus nerve firings in the rat
Authors: Shu Hai‐Feng , Wang Bai‐Ren , Wang Shi‐Rong , Yao Wei , Huang Hong‐Ping , Zhou Zhuan , Wang Xi , Fan Juan , Wang Ting , Ju Gong
Published: June 2007

Increasing evidence indicates that there exists a reciprocal communication between the immune system and the brain. Interleukin 1β (IL‐1β), a proinflammatory cytokine produced during immune challenge, is believed to be one of the mediators of immune‐to‐brain communication, but how it gets into the brain is unknown because of its large molecular weight and difficulty in crossing the blood–brain barrier. Our previous work has demonstrated that IL‐1 receptor type I is strongly expressed in the glomus cells of rat carotid body (CB), a well characterized polymodal chemoreceptive organ which serves not only for the detection of hypoxia, hypercapnia and acidity, but also for low temperature and blood glucose. The present study was designed to test whether IL‐1β could stimulate the CB glomus cells and alter the discharge properties in the carotid sinus nerve, the afferent nerve innervating the organ. The results from whole‐cell patch‐clamp recordings and calcium imaging showed that extracellular application of IL‐1β significantly decreased the outward potassium current and triggered a transient rise in [Ca2+]i in the cultured glomus cells of rat CB. Furthermore, by using extracellular recordings and pharmacological intervention, it was found that IL‐1β stimulation of the CB in the anaesthetized rat in vivo significantly increased the discharge rate in the carotid sinus nerve, most probably mediated by ATP release. This experiment provides evidence that the CB responds to cytokine stimulation and proposes the possibility that the CB might play a role in immune‐to‐brain communication.
A test of the opponent‐process theory of motivation using lesions that selectively block morphine reward
Authors: Vargas‐Perez Hector , Ting‐A‐Kee Ryan A. , Heinmiller Andrew , Sturgess Jessica E. , Van Der Kooy Derek
Published: June 2007

The opponent‐process theory of motivation postulates that motivational stimuli activate a rewarding process that is followed by an opposed aversive process in a homeostatic control mechanism. Thus, an acute injection of morphine in nondependent animals should evoke an acute rewarding response, followed by a later aversive response. Indeed, the tegmental pedunculopontine nucleus (TPP) mediates the rewarding effects of opiates in previously morphine‐naive animals, but not other unconditioned effects of opiates, or learning ability. The aversive opponent process for acute morphine reward was revealed using a place‐conditioning paradigm. The conditioned place aversion induced by 16‐h spontaneous morphine withdrawal from an acute morphine injection in nondependent rats was abolished by TPP lesions performed prior to drug experience. However, TPP‐lesioned rats did show conditioned aversions for an environment paired with the acute administration of the opioid antagonist naloxone, which blocks endogenous opioids. The results show that blocking the rewarding effects of morphine with TPP lesions also blocked the opponent aversive effects of acute morphine withdrawal in nondependent animals. Thus, this spontaneous withdrawal aversion (the opponent process) is induced by the acute rewarding effects of morphine and not by other unconditioned effects of morphine, the pharmacological effects of morphine or endogenous opioids being displaced from opiate receptors.
Lesions of the dorsal noradrenergic bundle impair attentional set‐shifting in the rat
Authors: Tait David S. , Brown Verity J. , Farovik Anja , Theobald David E. , Dalley Jeffrey W. , Robbins Trevor W.
Published: June 2007

Rats with medial prefrontal cortex (mPFC) lesions are impaired in attentional set‐shifting, when it is required to shift to a previously irrelevant perceptual dimension. The main source of noradrenergic input to the mPFC is from the locus coeruleus via the dorsal noradrenergic ascending bundle (DNAB). This study examined the effects of selective cortical noradrenaline depletion following 6‐hydroxydopamine‐induced lesions of the DNAB on attentional set‐shifting and other aspects of discrimination learning and performance. Rats learned to dig in baited bowls, and then acquired discriminations based on one of two aspects of a bowl − odour or digging medium. The task tested acquisition of novel discriminations (both intra‐ and extra‐dimensional) and reversal learning when contingencies were reversed with the same stimuli. At the conclusion of testing, the DNAB‐lesioned rats were shown to have a selective depletion of noradrenaline of ∼ 70% within the mPFC (cingulate and prelimbic cortex subregions), with no other significant changes in dopamine or 5‐hydroxytryptamine. Rats required more trials to learn new discriminations when attentional shifting was required [extra‐dimensional (ED)‐shift]. Rats with dorsal noradrenergic ascending bundle (DNAB) lesions were impaired in novel acquisitions when an ED‐shift was required, but were unimpaired in reversal learning and other aspects of discrimination learning, relative to controls. These data are consistent with other evidence implicating noradrenaline (NA) in attentional set‐shifting, and contrast with effects of manipulations of 5‐hydroxytryptamine (5‐HT) and acetylcholine within the medial prefrontal cortex (mPFC). The findings are also relevant to recent theorizing about the functions of the coeruleo‐cortical noradrenergic system.
Restorative respiratory pathways after partial cervical spinal cord injury: role of ipsilateral phrenic afferents
Authors: Vinit Stéphane , Stamegna Jean‐Claude , Boulenguez Pascale , Gauthier Patrick , Kastner Anne
Published: June 2007

After disruption of the descending respiratory pathways induced by unilateral cervical spinal cord injury (SCI) in rats, the inactivated ipsilateral (ipsi) phrenic nerve (PN) discharge may partially recover following some specific experimental procedures [such as contralateral (contra) phrenicotomy (Phx)]. This phrenic reactivation involves normally silent contra pathways decussating at the level of the phrenic nucleus, but the mechanisms of this crossed phrenic activation are still poorly understood. The present study investigates the contribution of sensory phrenic afferents to this process by comparing the acute effects of ipsi and contra Phx. We show that the phrenic discharge (recorded on intact PNs) was almost completely suppressed 0 h and 3 h after a lateral cervical SCI, but was already spontaneously reactivated after 1 week. This ipsi phrenic activity was enhanced immediately after contra Phx and was completely suppressed by an acute contra cervical section, indicating that it is triggered by crossed phrenic pathways located laterally in the contra spinal cord. Ipsi phrenic activity was also abolished immediately after ipsi Phx that interrupts phrenic sensory afferents, an effect prevented by prior acute ablation of the cervical dorsal root ganglia, indicating that crossed phrenic activation depends on excitatory phrenic sensory afferents but also putatively on inhibitory non‐phrenic afferents. On the basis of these data, we propose a new model for crossed phrenic activation after partial cervical injury, with an essential role played by ipsi‐activating phrenic sensory afferents.
Superior Vena Cava, Right Pulmonary Artery or Both: Real Time Two‐ and Three‐Dimensional Transthoracic Contrast Echocardiographic Identification of the Echo‐Free Space Posterior to the Ascending Aorta
Authors: Burri Manjula V. , Mahan Edward F. , Nanda Navin C. , Singh Anurag , Pothineni Koteswara R. , Yelamanchili Pridhvi , Panwar Sadik R.
Published: June 2007

We describe the usefulness of contrast echocardiography and live/real time three‐dimensional transthoracic echocardiography in characterizing the normal structures imaged posterior to the proximal ascending aorta as superior vena cava, right pulmonary artery, or both.
Improved Postprandial Response and Feeling of Satiety after Consumption of Low‐Calorie Muffins with Maltitol and High‐Amylose Corn Starch
Authors: Quílez J. , Bulló M. , Salas‐Salvadó J.
Published: June 2007

ABSTRACT:  The aim of this study was to determine the postprandial glucose (GR), insulin (IR), and triglyceride response as well as the satiety response (SR) to low‐calorie muffins (LCMs) and to compare them to those of conventional plain muffins (PMs) and bread. The LCMs had a lower fat content, their sugar was replaced by maltitol, and wheat flour was partially replaced by high‐amylose corn starch. We used bread as a reference to calculate glycemic (GI), insulinemic (II), and satiety indices (SI). Seven men and 7 women (33 ± 7.8 y; body mass index = 25.8 ± 2.9) were studied in a randomized crossover design and were given either bread, an LCM, or a PM during 3 different occasions. Plasma glucose, insulin, and triglyceride concentrations were periodically measured for 2 h after consumption. We used the same design in a separate assay to evaluate SR by means a subjective questionnaire. The results show that LCMs reduced the IR by 30% (P= 0.03) and lipemic response by 50% (P < 0.001) compared to PMs. GR was only found to be significantly different between bread and LCMs (52% lower in LCM; P= 0.03), with PMs in an intermediate position. The SR of LCMs is similar to bread and higher than PMs (191%; P= 0.02). We concluded that LCMs are a product with a very low GI with better postprandial and SRs than PMs. These metabolic properties are useful in normal and overweight persons. However, further research is needed on the effects of this type of products in individuals with impaired glucose tolerance.
Three‐Dimensional Observation of Connective Tissue of Bovine Masseter Muscle under Concentrate‐ and Roughage‐Fed Conditions by using Immunohistochemical/Confocal Laser‐Scanning Microscopic Methods
Authors: Nakamura Y.‐N. , Iwamoto H. , Etoh T. , Shiotsuka Y. , Yamaguchi T. , Ono Y. , Tabata S. , Nishimura S. , Gotoh T.
Published: June 2007

ABSTRACT:  We investigated changes in connective tissue components of masseter (MA) muscle in Japanese black heifers (n= 6) in concentrate‐ and roughage‐fed groups (groups C and R, respectively). Body weight, at slaughter, of experimental heifers in group C (272.3 ± 22.3 kg) was higher (P < 0.05) than that of group R (213.8 ± 27.5 kg). However, muscle weight and myofiber diameter (superficial and deep layers) of MA muscle did not differ between groups C and R. In contrast, total mastication duration of group R was longer (P < 0.05) than that of group C. MA muscle of groups C and R was composed only of type I myofiber. Using immunohistochemical/confocal laser‐scanning microscopy, type I collagen was observed mainly in perimysium, and type V and VI collagen were observed in perimysium and endomysium of both groups. Type IV collagen and laminin were observed only in the endomysium in both groups. However, type III collagen and fibronectin were strongly apparent in the perimysium and endomysium in group R. Connective tissue components in the perimysium of groups C and R were observed to form plate‐shaped layers. On the other hand, honeycomb‐shaped connective tissue components were seen in the endomysium‐surrounded muscle fibers. In particular, fibronectin was strongly observed in the perimysium and endomysium in group R. These results indicate that there are different developmental changes among connective tissue components in MA muscle in response to mastication. The immunohistochemical/confocal laser‐scanning microscopic method is useful to investigate the structural relationship among connective tissue components in skeletal muscle.
Phenolic Acid Content and Composition in Leaves and Roots of Common Commercial Sweetpotato (Ipomea batatas L.) Cultivars in the United States
Authors: Truong V.‐D. , McFeeters R.F. , Thompson R.T. , Dean L.L. , Shofran B.
Published: June 2007

ABSTRACT:  Phenolic acids in commercially important sweet potato cultivars grown in the United States were analyzed using reversed‐phase high‐performance liquid chromatography (HPLC). Caffeic acid, chlorogenic acid, 4,5‐di‐O‐caffeoylquinic acid, 3,5‐di‐O‐caffeoylquinic acid, and 3,4‐di‐O‐caffeoylquinic acid were well separated with an isocratic elution in less than 25 min compared to about 120 min for analyzing and re‐equilibrating the column with a gradient method. The isocratic elution order of these caffeoylquinic acid derivatives was confirmed by LC‐MS/MS. Chlorogenic acid was the highest in root tissues, while 3,5‐di‐O‐caffeoylquinic acid and/or 4,5‐di‐O‐caffeoylquinic acid were predominant in the leaves. Steam cooking resulted in statistically nonsignificant increases in the concentration of total phenolics and all the individual phenolic acids identified. Sweetpotato leaves had the highest phenolic acid content followed by the peel, whole root, and flesh tissues. However, there was no significant difference in the total phenolic content and antioxidant activity between purees made from the whole and peeled sweet potatoes.
A Comparison of Protocols and Observer Precision for Measuring Physical Stream Attributes1
Authors: Whitacre Heath W. , Roper Brett B. , Kershner Jeffrey L.
Published: June 2007

Abstract: Stream monitoring programs commonly measure physical attributes to assess the effect of land management on stream habitat. Variability associated with the measurement of these attributes has been linked to a number of factors, but few studies have evaluated variability due to differences in protocols. We compared six protocols, five used by the U.S. Department of Agriculture Forest Service and one by the U.S. Environmental Protection Agency, on six streams in Oregon and Idaho to determine whether differences in protocol affect values for 10 physical stream attributes. Results from Oregon and Idaho were combined for groups participating in both states, with significant differences in attribute means for 9 out of the 10 stream attributes. Significant differences occurred in 5 of 10 in Idaho, and 10 of 10 in Oregon. Coefficients of variation, signal‐to‐noise ratio, and root mean square error were used to evaluate measurement precision. There were differences among protocols for all attributes when states were analyzed separately and as a combined dataset. Measurement differences were influenced by choice of instruments, measurement method, measurement location, attribute definitions, and training approach. Comparison of data gathered by observers using different protocols will be difficult unless a core set of protocols for commonly measured stream attributes can be standardized among monitoring programs.
Induction of CD4-Independent E7-Specific CD8
+
Memory Response by Heat Shock Fusion Protein
Authors: Liu Hongwei , Wu Bill H. , Rowse Gerry J. , Emtage Peter C. R.
Published: June 2007

Infection with human papillomavirus type 16 (HPV16) is strongly associated with a number of disease states, of which cervical and anal cancers represent the most drastic endpoints. Induction of T-cell-mediated immunity, particularly cytotoxic T lymphocytes (CTL), is important in eradication of HPV-induced lesions. Studies have shown that heat shock protein fusion proteins are capable of inducing potent antigen-specific CTL activity in experimental animal models. In addition, E7-expressing tumors in C57BL/6 mice can be eradicated by treatment with HspE7, an Hsp fusion protein composed of
Mycobacterium bovis
BCG Hsp65 linked to E7 protein of HPV16. More importantly, HspE7 has also displayed significant clinical benefit in phase II clinical trials for the immunotherapy of HPV-related diseases. To delineate the mechanisms underlying the therapeutic effects of HspE7, we investigated the capability of HspE7 to induce antigen-specific protective immunity. Here, we demonstrate that HspE7 primes potent E7-specific CD8
+
T cells with cytolytic and cytokine secretion activities. These CD8
+
T cells can differentiate into memory T cells with effector functions in the absence of CD4
+
T-cell help. The HspE7-induced memory CD8
+
T cells persist for at least 17 weeks and confer protection against E7-positive murine tumor cell challenge. These results indicate that HspE7 is a promising immunotherapeutic agent for treating HPV-related disease. Moreover, the ability of HspE7 to induce memory CD8
+
T cells in the absence of CD4
+
help indicates that HspE7 fusion protein may have activity in individuals with compromised CD4
+
functions, such as those with invasive cancer and/or human immunodeficiency virus infection.
Antibodies Generated in Cats by a Lipopeptide Reproducing the Membrane-Proximal External Region of the Feline Immunodeficiency Virus Transmembrane Enhance Virus Infectivity
Authors: Giannecchini Simone , D'Ursi Anna Maria , Esposito Cinzia , Scrima Mario , Zabogli Elisa , Freer Giulia , Rovero Paolo , Bendinelli Mauro
Published: June 2007

The immunogenicity of a lipoylated peptide (lipo-P59) reproducing the membrane-proximal external region (MPER) of the transmembrane glycoprotein of feline immunodeficiency virus (FIV) was investigated with cats. In the attempt to mimic the context in which MPER is located within intact virions, lipo-P59 was administered in association with membrane-like micelles. Analyses showed that in this milieu, lipo-P59 had a remarkable propensity to be positioned at the membrane interface, displayed a large number of ordered structures folded in turn helices, and was as active as lipo-P59 alone at inhibiting FIV infectivity in vitro. The antibodies developed differed from the ones previously obtained by immunizing cats with the nonlipoylated version of the peptide (G. Freer, S. Giannecchini, A. Tissot, M. F. Bachmann, P. Rovero, P. F. Serres, and M. Bendinelli, Virology 322:360-369, 2004) in epitope specificity and in the fact that they bound FIV virions. However, they too lacked virus-neutralizing activity and actually enhanced FIV infectivity for lymphoid cell cultures. It is concluded that the use of MPER-reproducing oligopeptides is not a viable approach for vaccinating against FIV.
Characterization of
Aerococcus viridans
Isolates from Swine Clinical Specimens
Authors: Martín V. , Vela A. I. , Gilbert M. , Cebolla J. , Goyache J. , Domínguez L. , Fernández-Garayzábal J. F.
Published: June 2007

We present here the biochemical and genetic characterization and antimicrobial susceptibility of 58 isolates of
Aerococcus viridans
isolated in pure culture from different clinical specimens of normally sterile body sites of pigs.
A. viridans
isolates were commonly susceptible to β-lactam antimicrobials and exhibited a great genetic heterogeneity as determined by pulsed-field gel electrophoresis typing. The results indicate that
A. viridans
might be included in the list of possible etiological agents causing disease in pigs.
Prevalence of Melioidosis in the Er-Ren River Basin, Taiwan: Implications for Transmission
Authors: Su Hsun-Pi , Yang Hsiao-Wei , Chen Ya-Lei , Ferng Tien-Lin , Chou Yu-Ling , Chung Tung-Ching , Chen Chang-Hsun , Chiang Chuen-Sheue , Kuan Mei-Mei , Lin Hsi-Hsun , Chen Yao-Shen
Published: June 2007

An increase in melioidosis cases compared to other areas in Taiwan was observed in the Er-Ren River Basin, southwestern Taiwan, from November 2001 to August 2006. The objective of this study was to determine the association between the level of exposure to
Burkholderia pseudomallei
and the incidence rate of melioidosis and to survey the transmission modes of
B. pseudomallei
in the Er-Ren River Basin. The serosurveillance of melioidosis gave seropositivity rates of 36.6%, 21.6%, and 10.9%, respectively, for residents in regions A, B, and C within the Er-Ren Basin area. Culture and PCR-based detection of
B. pseudomallei
from soil demonstrated that the geographical distribution of this bacterium was confined to a particular site in region B. The distribution of seropositive titers was significantly associated with the incidence rate of melioidosis (120, 68, or 36 incidence cases per 100,000 population in region A, B, or C in 2005), whereas it did not correlate with the geographical distribution of
B. pseudomallei
within the soil. A survey of transmission modes showed that residents with seropositivity were linked to factors such as having confronted flooding and having walked barefoot on soil, which are potential risk factors associated with exposure to
B. pseudomallei
. Our findings indicated that the Er-Ren River Basin in Taiwan has the potential to become a high-prevalence area for melioidosis. This is the first report that documents a high prevalence of melioidosis in an area north of latitude 20°N.
Reclassification of Phenotypically Identified
Staphylococcus intermedius
Strains
Authors: Sasaki Takashi , Kikuchi Ken , Tanaka Yoshikazu , Takahashi Namiko , Kamata Shinichi , Hiramatsu Keiichi
Published: June 2007

To reclassify phenotypically identified
Staphylococcus intermedius
strains, which might include true
S. intermedius
strains and novel species such as
Staphylococcus pseudintermedius
and
Staphylococcus delphini
, we analyzed molecular phylogenies and phenotypic characteristics of 117
S. intermedius
group (SIG) strains tentatively identified as being
S. intermedius
by the Rapid ID32 Staph assay. From phylogenetic analyses of
sodA
and
hsp60
sequences, the SIG strains were divided into three clusters, which belonged to
S. pseudintermedius
LMG 22219
T
,
S. intermedius
ATCC 29663
T
, and
S. delphini
LMG 22190
T
. All the SIG strains from dogs, cats, and humans were identified as being
S. pseudintermedius
. The wild pigeon strains, except one, were identified as being
S. intermedius
, and strains from all domestic pigeons, one wild pigeon, horses, and a mink were identified as being
S. delphini
. In addition, a phylogenetic analysis of
nuc
genes revealed that
S. delphini
strains were divided into two clusters: one was the cluster (
S. delphini
group A) that belonged to
S. delphini
LMG 22190
T
, and the other was the cluster (
S. delphini
group B) that was more related to
S. pseudintermedius
LMG 22219
T
than
S. delphini
LMG 22190
T
. The DNA-DNA hybridization results showed that
S. delphini
group B strains were distinguished from
S. delphini
group A,
S. intermedius
, and
S. pseudintermedius
strains.
S. intermedius
is distinguishable from
S. pseudintermedius
or
S. delphini
by positive arginine dihydrolase and acid production from β-gentiobiose and
d
-mannitol. However, phenotypical characteristics to differentiate
S. delphini
group A,
S. delphini
group B, and
S. pseudintermedius
were not found. In conclusion, SIG strains were reclassified into four clusters with three established and one probably novel species.
A Baculovirus-Expressed Dicistrovirus That Is Infectious to Aphids
Authors: Pal Narinder , Boyapalle Sandhya , Beckett Randy , Miller W. Allen , Bonning Bryony C.
Published: June 2007

Detailed investigation of virus replication is facilitated by the construction of a full-length infectious clone of the viral genome. To date, this has not been achieved for members of the family
Dicistroviridae
. Here we demonstrate the construction of a baculovirus that expresses a dicistrovirus that is infectious in its natural host. We inserted a full-length cDNA clone of the genomic RNA of the dicistrovirus
Rhopalosiphum padi virus
(RhPV) into a baculovirus expression vector. Virus particles containing RhPV RNA accumulated in the nuclei of baculovirus-infected Sf21 cells expressing the recombinant RhPV clone. These virus particles were infectious in
R. padi
, a ubiquitous aphid vector of major cereal viruses. The recombinant virus was transmitted efficiently between aphids, despite the presence of 119 and 210 vector-derived bases that were stably maintained at the 5′ and 3′ ends, respectively, of the RhPV genome. The maintenance of such a nonviral sequence was surprising considering that most RNA viruses tolerate few nonviral bases beyond their natural termini. The use of a baculovirus to express a small RNA virus opens avenues for investigating replication of dicistroviruses and may allow large-scale production of these viruses for use as biopesticides.
Identification of HI-Like Loop in CELO Adenovirus Fiber for Incorporation of Receptor Binding Motifs
Authors: Logunov Denis Y. , Zubkova Olga V. , Karyagina-Zhulina Anna S. , Shuvalova Eugenia A. , Karpov Andrei P. , Shmarov Maxim M. , Tutykhina Irina L. , Alyapkina Yulia S. , Grezina Natalia M. , Zinovieva Natalia A. , Ernst Lev K. , Gintsburg Alexsandr L. , Naroditsky Boris S.
Published: June 2007

Vectors based on the chicken embryo lethal orphan (CELO) avian adenovirus (Ad) have two attractive properties for gene transfer applications: resistance to preformed immune responses to human Ads and the ability to grow in chicken embryos, allowing low-cost production of recombinant viruses. However, a major limitation of this technology is that CELO vectors demonstrate decreased efficiency of gene transfer into cells expressing low levels of the coxsackie-Ad receptor (CAR). In order to improve the efficacy of gene transfer into CAR-deficient cells, we modified viral tropism via genetic alteration of the CELO fiber 1 protein. The αv integrin-binding motif (RGD) was incorporated at two different sites of the fiber 1 knob domain, within an HI-like loop that we identified and at the C terminus. Recombinant fiber-modified CELO viruses were constructed containing secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein genes as reporter genes. Our data show that insertion of the RGD motif within the HI-like loop of the fiber resulted in significant enhancement of gene transfer into CAR-negative and CAR-deficient cells. In contrast, CELO vectors containing the RGD motif at the fiber 1 C terminus showed reduced transduction of all cell lines. CELO viruses modified with RGD at the HI-like loop transduced the SEAP reporter gene into rabbit mammary gland cells in vivo with an efficiency significantly greater than that of unmodified CELO vector and similar to that of Ad type 5 vector. These results illustrate the potential for efficient CELO-mediated gene transfer into a broad range of cell types through modification of the identified HI-like loop of the fiber 1 protein.
Characterization of the Sequence Element Directing Translation Reinitiation in RNA of the Calicivirus Rabbit Hemorrhagic Disease Virus
Authors: Meyers Gregor
Published: June 2007

The calicivirus minor capsid protein VP2 is expressed via reinitiation of protein synthesis after termination of translation of the preceding VP1 gene. A sequence element of about 80 nucleotides denoted “termination upstream ribosomal binding site” (TURBS) (25) is crucial for reinitiation. Deletion mapping in the TURBS of a rabbit calicivirus identified two short sequence motifs that were crucial for VP2 expression. Motif 1 is conserved among caliciviruses and is complementary to a sequence in the 18S rRNA. Single-residue exchanges in this motif severely impaired reinitiation when they affected the putative rRNA binding, whereas an exchange preserving complementarity had only a minor effect. Single exchanges in motif 2 were rather well tolerated, but the introduction of double exchanges almost blocked VP2 expression. In contrast, the deletion analyses showed that the RNA between the two motifs is of minor importance. The distance between motif 2 and the start site was found to be important, since deletions of increasing length in this sequence or upstream positioning of the start codon reduced VP2 expression stepwise to low levels, whereas multiple-nucleotide exchanges in this region were tolerated. The low flexibility of the arrangement of TURBS motif 2 and the start codon stand in marked contrast to the requirements with regard to the location of the stop codon of the preceding VP1 gene, which could be moved far downstream with continuous reduction, but without loss, of VP2 translation. The sequence mapping resulted in a refined model of the reinitiation mechanism leading to VP2 expression.
In Vivo and In Vitro Intragenomic Rearrangement of TT Viruses
Authors: Leppik Ludmila , Gunst Karin , Lehtinen Matti , Dillner Joakim , Streker Karin , de Villiers Ethel-Michele
Published: June 2007

The in vitro replication of the Torque teno virus (TT virus) tth8 full-length genome and particle formation in a Hodgkin's lymphoma-derived cell line after transfection with cloned viral DNA were demonstrated. Analyses of the transcription patterns of tth8 and tth7 TT virus isolates in a number of lymphoma and T-cell leukemia cell lines indicated differential additional splicing events and intragenomic rearrangement generating open reading frames which could not be deducted from the genomic sequence. We also demonstrated the presence of rearranged TT virus genomes in vivo in sera taken from pregnant mothers whose children later developed childhood leukemia, as well as sera from control mothers. Control experiments using religated cloned genomic tth8 DNA mixed with cellular DNA did not result in such subviral molecules. These subviral isolates ranged from 172 bp to full-length TT virus genomes. Possible in vivo selection for specific rearranged molecules was indicated by the presence of one isolate (561 bp) in 11 serum samples. It remains to be clarified whether selected rearranged subviral components resulting from specific TT virus types may contribute to the initiation of disease. These data demonstrate new features of TT viruses suggesting possible similarities to plant viruses of the family
Geminiviridae
, as well as raise questions about the documented plurality and diversity of anelloviruses.
A New Generation of Modified Live-Attenuated Avian Influenza Viruses Using a Two-Strategy Combination as Potential Vaccine Candidates
Authors: Song Haichen , Nieto Gloria Ramirez , Perez Daniel R.
Published: June 2007

In light of the recurrent outbreaks of low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI), there is a pressing need for the development of vaccines that allow rapid mass vaccination. In this study, we introduced by reverse genetics temperature-sensitive mutations in the PB1 and PB2 genes of an avian influenza virus, A/Guinea Fowl/Hong Kong/WF10/99 (H9N2) (WF10). Further genetic modifications were introduced into the PB1 gene to enhance the attenuated (att) phenotype of the virus in vivo. Using theattWF10 as a backbone, we substituted neuraminidase (NA) for hemagglutinin (HA) for vaccine purposes. In chickens, a vaccination scheme consisting of a single dose of anattH7N2 vaccine virus at 2 weeks of age and subsequent challenge with the wild-type H7N2 LPAI virus resulted in complete protection. We further extended our vaccination strategy against the HPAI H5N1. In this case, we reconstituted anattH5N1 vaccine virus, whose HA and NA genes were derived from an Asian H5N1 virus. A single-dose immunization in ovo with theattH5N1 vaccine virus in 18-day-old chicken embryos resulted in more than 60% protection for 4-week-old chickens and 100% protection for 9- to 12-week-old chickens. Boosting at 2 weeks posthatching provided 100% protection against challenge with the HPAI H5N1 virus for chickens as young as 4 weeks old, with undetectable virus shedding postchallenge. Our results highlight the potential of liveattavian influenza vaccines for mass vaccination in poultry.