Role of the XylA gene, encoding a cell wall degrading enzyme, during common wheat, durum wheat and barley colonization by Fusarium graminearum.

Fusarium graminearum is the main causal agent of fusarium head blight (FHB) of wheat and barley. This filamentous fungus is able to produce hydrolytic enzymes, such as xylanases, that cause cell wall degradation, permitting host colonization. This study investigated the role of the F. graminearum XylA (FGSG_10999) gene during infection, using a knockout mutant in strain CS3005. Assays were carried out on common wheat, durum wheat and barley to compare virulence of a XylA knockout to that of wild type strain. These assays were conducted on wheat and barley seedling roots, seedling stem bases and heads. Furthermore, additional in vitro experiments were conducted to investigate the role of XylA gene in the utilisation of D-xylose, the main component of cereals cell wall. In planta assays showed the importance of XylA gene for F. graminearum virulence towards its main hosts. A positive correlation between symptom incidence and fungal biomass development was also observed for both the wild type and the knockout strains. Finally, gene expression studies performed in a liquid medium enriched with D-xylose, a known xylanase inducer in other fungi, showed that the absence of the gene in the FGSG_10999 locus was not compensated by two other F. graminearum xylanase encoding genes analysed (loci FGSG_06445 and FGSG_11478).

Identification of Heparan-Sulfate Rich Cells in the Loose Connective Tissues of the Axolotl (Ambystoma mexicanum) with the Potential to Mediate Growth Factor Signaling during Regeneration.

Limb regeneration is the outcome of a complex sequence of events that are mediated by interactions between cells derived from the tissues of the amputated stump. Early in regeneration, these interactions are mediated by growth factor/morphogen signaling associated with nerves and the wound epithelium. One shared property of these proregenerative signaling molecules is that their activity is dependent on interactions with sulfated glycosaminoglycans (GAGs), heparan sulfate proteoglycan (HSPG) in particular, in the extracellular matrix (ECM). We hypothesized that there are cells in the axolotl that synthesize specific HSPGs that control growth factor signaling in time and space. In this study we have identified a subpopulation of cells within the ECM of axolotl skin that express high levels of sulfated GAGs on their cell surface. These cells are dispersed in a grid-like pattern throughout the dermis as well as the loose connective tissues that surround the tissues of the limb. These cells alter their morphology during regeneration, and are candidates for being a subpopulation of connective tissue cells that function as the cells required for pattern-formation during regeneration. Given their high level of HSPG expression, their stellate morphology, and their distribution throughout the loose connective tissues, we refer to these as the positional information GRID (Groups that are Regenerative, Interspersed and Dendritic) cells. In addition, we have identified cells that stain for high levels of expression of sulfated GAGs in mouse limb connective tissue that could have an equivalent function to GRID cells in the axolotl. The identification of GRID cells may have important implications for work in the area of Regenerative Engineering.

Neurotrophic regulation of epidermal dedifferentiation during wound healing and limb regeneration in the axolotl (Ambystoma mexicanum).

Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control blastema morphogenesis and limb regeneration. In the present study, we have utilized the ALM to identity the buttonhead-like zinc-finger transcription factor, Sp9, as being involved in the formation of the regeneration epithelium. Sp9 expression is induced in basal keratinocytes of the apical blastema epithelium in a pattern that is comparable to its expression in developing limb buds, and it thus is an important marker for dedifferentiation of the epidermis. Induction of Sp9 expression is nerve-dependent, and we have identified KGF as an endogenous nerve factor that induces expression of Sp9 in the regeneration epithelium.

Membrane junctions in Xenopus eggs: their distribution suggests a role in calcium regulation.

We have observed the presence of membrane junctions formed between the plasma membrane and cortical endoplasmic reticulum of mature, unactivated eggs of xenopus laevis. The parallel, paired membranes of the junction are separated by a 10-mn gap within which electron-dense material is present. This material occurs in patches with an average center-to-center distance of approximately 30 nm. These junctions are rare in immature (but fully grown) oocytes (approximately 2 percent of the plasma membrane is associated with junctions) and increase dramatically during progesterone-induced maturation. Junctions in the mature, unactivated egg are two to three times more abundant in the animal hemisphere (25-30 percent of the plasma membrane associated with junction) as compared with the vegetal hemisphere (10-15 percent). Junction density decreases rapidly to values characteristic of immature oocytes in response to egg activation. The plasma membrane-ER junctions of xenopus eggs are strikingly similar in structure to membrane junctions in muscle cells thought to be essential in the triggering of intracellular calcium release from the sarcoplasmic reticulum. In addition, the junctions' distinctive, animal-vegetal polarity of distribution, their dramatic appearance during maturation, and their disapperance during activation are correlated with previously documented patterns of calcium-mediated events in anuran eggs. We discuss several lines of evidence supporting the hypothesis that these junctions in xenopus eggs are sites that transduce extracellular events into intracellular calcium release during fertilization and activation of development.

Conserved vertebrate chromosome segments in the large salamander genome.

Urodele amphibians (salamanders) are important models for embryological, physiological, and natural history research and are also a biomedically important group because they are the only vertebrates capable of regenerating entire organ systems. To enhance the utility of salamanders for biomedical research and for understanding genome evolution, genetic linkage analysis was used to identify chromosome segments that are homologous between ambystomatid salamanders and distantly related vertebrate model organisms. A total of 347 loci (AFLPs, RAPDs, and protein-coding loci) were mapped using an interspecific meiotic mapping panel (Ambystoma mexicanum and A. tigrinum tigrinum; family Ambystomatidae). Genome size in Ambystoma was estimated to be 7291 cM, the largest linkage map estimate reported for any organism. However, the relatively large size of the salamander genome did not hinder efforts to map and identify conserved syntenies from a small sample of 24 protein-coding loci. Chromosomal segments that are conserved between fishes and mammals are also conserved in these salamanders. Thus, comparative gene mapping appears to be an efficient strategy for identifying orthologous loci between ambystomatid salamanders and genomically well-characterized vertebrate model organisms.

Molecular mechanisms in the control of limb regeneration: the role of homeobox genes.

Axolotls are unique among vertebrates in their ability to regenerate lost appendages as adults. They provide the opportunity to study the mechanism of regeneration in vertebrates and are an inspiration to pursue the goal of appendage regeneration in humans. In this article, we review data on the role of homeobox-containing genes in the regulation of limb regeneration. As a group, these genes are important in pattern formation in the primary body axis, developing limbs and regenerating limbs. To date, a total of 22 homeobox genes have been identified as being expressed in regenerating limbs. Nearly all of these are also expressed during limb regeneration, further supporting the view that limb development and regeneration involve similar regulatory mechanisms. Our recent results on the expression of HoxA genes demonstrate that once a blastema has formed, subsequent outgrowth and pattern formation are similar to those of limb development. In contrast to developing limbs, reexpression of the HoxA genes in regeneration occurs by a non-colinear mechanism that likely is related to the necessity of mature limb cells to undergo dedifferentiation in order to give rise to the blastema. These studies also indicate that the pattern is respecified by a distal-first mechanism during regeneration in contrast to the apparent proximal-to-distal sequence observed in developing limbs. Expression of the HoxA genes is altered coordinately in response to retinoic acid in a manner consistent with the transformation of a distal blastema to a proximal blastema. Given the recent increase in studies of the molecules involved in regeneration, it is likely that many of the functionally important regeneration genes will be identified and characterized in the near future.

Cyclic changes in fine structure of the epithelium lining the ovary of the viviparous teleost, Cymatogaster aggregata (Perciformes: Embiotocidae).

The most striking morphological feature of the ovarian epithelium of Cymatogaster is the presence of intercellular dilations during much of the year. These dilations increase markedly in volume during the several months prior to ovulation and fertilization, and decrease in volume during the months of embryogenesis and gestation. The epithelium then returns to its initial, relatively undifferentiated state. The extracellular material within the dilations likely is synthesized in the cells adjacent to or within the dilations. Apparently most of this material is released into the ovarian lumen when the apicolateral margins of adjacent epithelial cells pull apart; possibly it serves as nutrient for developing embryos. In addition to supporting embryogenesis, the ovarian epithelium also apparently is involved in sperm storage. Sperm are maintained within pockets in the ovarian epithelium for the several months between insemination and fertilization. The cells lining the sperm pocket do not develop the intercellular dilations characteristic of most of the ovarian epithelium, and sperm remain associated only with the sperm pocket cells.

Biochemical comparison of plaque fluid on tooth and acrylic surfaces during a sucrose challenge.

Previous studies have investigated variations in dental plaque fluid composition within a single mouth after a sucrose exposure. The purpose of this study was to determine a potential source of calcium and phosphorus in plaque by comparing the pH, calcium and phosphorus concentrations in plaque fluid obtained from an acrylic appliance with samples taken from supragingival tooth surfaces within the same individual after a sucrose challenge. Separate plaque samples from 14 individuals were collected from an acrylic appliance or tooth surfaces within same individual before and 15 min after a 20% sucrose rinse. Each plaque sample was centrifuged and nanolitre samples of plaque fluid were analysed for pH with a pH microelectrode, for total calcium concentration by atomic absorption in a graphite furnace, and for phosphorus concentration by spectrophotometry. There was an increase in the calcium and phosphorus concentration in the plaque after the sucrose challenge and a significant increase in calcium and phosphorus concentrations in the plaque taken from the teeth compared to the acrylic surfaces. The results indicate that the increased total calcium and phosphorus in plaque during a sucrose challenge is probably derived from the demineralization of enamel or extracellular demineralized components.

Attitudinal factors influencing mouthguard utilization.

From the introduction of a protective mouthpiece in the sport of boxing, the evidence for the importance of this piece of athletic equipment in injury prevention has grown. Yet, despite this knowledge, only five amateur sports and one professional sport have regulations requiring the use of mouthguards. Even in the sports that require their use, compliance is not universal. Attitudes of coaches, officials, parents, and players about wearing mouthguards all influence their usage. Studies of the attitudes of these groups reveal that coaches are perceived as the individuals with the most impact on whether or not players wear mouthguards. There is reluctance among college football officials to enforce mouthguard violations that they believe are inappropriate. Parents see themselves as having responsibility in determining mouthguard use; however, their views about when and for whom mouthguards are necessary reveal a lack of complete understanding of the benefits. Resistance on the part of players stems from the physical characteristics of the mouthguard, interference with breathing and speech, and the effect on the players' image. Education on the effectiveness of properly fitted mouthguards for injury prevention, information on the risk for injury, availability of more comfortable and appealing mouthguards, and development of an approach for expanding regulations are all tools that can lead to the development of more positive attitudes and increased usage.

Psychosocial issues in women's oral health.

Oral health is a significant issue for women because of its association with overall health and quality of life. There is growing evidence that the casual factors and consequences of oral disease are not only physical, but are also social and psychologic. As the importance and implications of the psychosocial factors become better understood, a more comprehensive model for treatment is developing. Accepting this role can lead dentists to a more multidisciplinary approach to successful treatment for their female patients.

Infection control procedures used in conjunction with computed dental radiography.

Infection control guidelines for dental radiography have been modified since 1986, when the American Dental Association and the Centers for Disease Control and Prevention supported the concept of "universal blood and body fluid precautions." With the introduction of computed digital radiography, hardware manufacturers recommend that alternative infection control techniques are necessary to prevent potential damage to the digital x-ray sensors placed inside the patient's mouth. Thirty first-year dental hygiene students were asked to insert and remove a Schick CDR number 2 size intraoral digital x-ray sensor into modified Rinn XCP bitewing bite blocks and a modified Rinn Snap-a-ray five times with each of the recommended infection control covers. Reduced rates of cross contamination are possible if the plastic barrier envelope has an additional latex finger cot stretched over it and the x-ray sensor. Sole usage of a latex finger cot will result in a reduced incidence of contamination, but still not to acceptable levels. However, a plastic barrier envelope placed over the x-ray sensor and over the modified XCP bite block together or a covered sensor in a Snap-a-ray under normal conditions does not result in a perforation and is least likely to result in cross contamination.

The effect of four-week administration of amitriptyline on sleep bruxism. A double-blind crossover clinical study.

The purpose of this study was to evaluate the effect of a tricyclic antidepressant (amitriptyline) on nocturnal masseteric activity and duration of sleep in bruxists. Using a randomized, double blind, crossover design, ten females (mean age 39 yrs, +/- sd seven yrs) received active (amitriptyline 25 mg/night) and inactive (placebo 25 mg/night) medication, over a period of four weeks each. A portable EMG integrator recorded the nocturnal, unilateral, and cumulative myoelectrical activity (microV/min of sleep) of the masseter muscle during the fourth and eighth weeks of the study. The results showed that amitriptyline did not significantly decrease the mean EMG activity (df = 9, alpha = 0.05, paired-t = 0.892, p = 0.3964), nor did it significantly increase the duration of sleep (df = 9, alpha = 0.05, paired-t = 2.140, p = 0.061). The results of this study do not support the administration of 25 mg of amitriptyline per night over a period of four weeks for the management of sleep bruxism.

Environmentally induced limb malformations in mink frogs (Rana septentrionalis).

In recent years, there has been an increase in the incidence of frog deformities throughout many of the northern states of North America. The most readily noticed malformations involve the hindlimbs of peri-metamorphic animals. We have analyzed skeletal preparations of metamorphosing mink frogs (Rana septentrionalis) collected from a site in Minnesota, in order to develop a better understanding of the possible causes. In this paper we describe the categories of abnormalities found at this site. The spectrum of deformities includes missing limbs, truncated limbs, extra limbs (including extra pelvic girdles), and skin webbings. We also describe a newly recognized malformation of the proximal-distal limb axis, a bony triangle. In this abnormality, the proximal and distal ends of the bone are adjacent to one another forming the base of a triangle. The shaft of the bone is bent double and protrudes laterally, the midpoint of the bone forming the apex of the triangle. In this paper we consider several recently proposed explanations for the recent outbreak of amphibian deformities. Based on our analysis, we conclude that the spectrum of abnormalities seen in these frogs is remarkably similar to the range of abnormalities that has been reported as a result of exposure of developing vertebrates to exogenous retinoids. Given the potential implications of this possibility for the welfare of humans as well as wildlife, further studies are needed to determine whether environmental retinoids are responsible for the frog deformities at the site we have examined.

Organization of positional information in the axolotl limb.

We have used the phenomenon of position-dependent growth stimulation, brought about by the confrontation of cells with dissimilar positional values, to reveal the organization of positional information in the center of the upper and lower arms of axolotls. When either humerus or radius was transplanted into either dorsal or posterior positions, extra growth leading to the formation of supernumerary digits occurred following amputation through the graft. However, transplants of humerus or radius into anterior or ventral positions did not lead to the formation of any additional digits. The ulna by contrast was capable of stimulating supernumerary digit formation when transplanted into anterior, posterior, dorsal, or ventral positions. We interpret these results to indicate that the humerus and radius are surrounded by symmetrically arranged anterior and ventral positional values, whereas the ulna is surrounded by a complete asymmetrical set of angular positional values. We use our proposed arrangement for the positional information in the limb center to explain a number of previous experimental findings. In addition, we provide an explanation, in terms of the underlying positional information, for the structural and developmental relationships between the different skeletal elements of the vertebrate limb, and in particular for the anatomical pattern known as Gregory's pyramid.

Retinoic acid, local cell-cell interactions, and pattern formation in vertebrate limbs.

Retinoic acid (RA), a derivative of vitamin A, has remarkable effects on developing and regenerating limbs. These effects include teratogenesis, arising from RA's ability to inhibit growth and pattern formation. They also include pattern duplication, arising as a result of the stimulation of additional growth and pattern formation. In this review we present evidence that the diverse effects of RA are consistent with a singular, underlying explanation. We propose that in all cases exogenously applied RA causes the positional information of pattern formation-competent cells to be reset to a value that is posterior-ventral-proximal with respect to the limb. The diversity of outcomes can be seen as a product of the mode of application of exogenous RA (global versus local) coupled with the unifying concept that growth and pattern formation in both limb development and limb regeneration are controlled by local cell-cell interactions, as formulated in the polar coordinate model. We explore the possibility that the major role of endogenous RA in limb development is in the establishment of the limb field rather than as a diffusible morphogen that specifies graded positional information across the limb as previously proposed. Finally, we interpret the results of the recent finding that RA can turn tail regenerates into limbs, as evidence that intercalary interactions may also be involved in the formation of the primary body axis.

Cell cycle length affects gene expression and pattern formation in limbs.

The relationship between growth and pattern specification during development remains elusive. Some molecules known to function as growth factors are also potent agents of pattern formation. This raises the possibility that growth factors could act in pattern formation via an effect on the cell cycle. We have tested the significance of the length of the cell cycle for gene expression and pattern formation in developing chick limb buds by locally slowing the cell cycle. When anterior cell cycles are lengthened by reversible inhibition of DNA replication or by other means, some genes characteristic of the posterior polarizing region are expressed, and digit duplication is observed. Conversely, when posterior cell cycles are slowed, expression of some posterior-specific genes is inhibited, but the pattern is normal. These results indicate that control of the length of the cell cycle could play a primary role in pattern formation by influencing the complement of genes expressed in a particular region of the embryo.

Patient satisfaction in a dental school.

PURPOSE: The objective of this study was to measure patients' satisfaction with the facility, services and treatment received at a dental school clinic. METHOD: The 31-item questionnaire consisted of demographic items and items in a 5-point Likert-type format addressing issues such as security, satisfaction with the facility, helpfulness of the staff, progress of treatment, fees, universal precautions taken, quality of care and treatment with dignity and compassion. The questionnaire also asked whether the patient would recommend the school to others seeking dental care. In 1997, patients completed the survey in the clinic waiting room prior to their dental appointment for that day. The survey was conducted over 3 months until 500 patients had participated, representing about 16% of the active patient population. %s of responses were computed and cross tabulations were used to compare responses based on demographics. RESULTS: The most frequently reported reason for wanting to be a patient at the dental school was low cost (67%) followed by up-to-date care (19%). 45% of patients indicated that a friend, neighbour or relative referred them, followed by 34% indicating referral by a dentist. More than 90% responded positively to the items concerning issues related to treatment except for satisfaction with progress of treatment and comparison of care received at the school to care elsewhere. For these 2 items, at least 80% responded positively. 99% indicated they would recommend the dental school to others seeking dental care. CONCLUSION: Results indicate that the vast majority of the patients surveyed are satisfied with the facility, services and treatment received. Comments that addressed areas of concern included the length of time to get an appointment and the length of time the appointment took, and it was recommended that the process of treatment be expedited.

Expression of Msx-2 during development, regeneration, and wound healing in axolotl limbs.

Msx genes are transcription factors that are expressed during embryogenesis of developing appendages in regions of epithelial-mesenchymal interactions. Various lines of evidence indicate that these genes function to maintain embryonic tissues in an undifferentiated, proliferative state. We have identified the axolotl homolog of Msx-2, and investigated its expression during limb development, limb regeneration, and wound healing. As in limb buds of higher vertebrates, axolotl Msx-2 is expressed in the apical epidermis and mesenchyme; however, its expression domain is more extensive, reflecting the broader region of the apical epidermal cap in amphibians. Msx-2 expression is downregulated at late stages of limb development, but is reexpressed within one hour after limb amputation. Msx-2 is also reexpressed during wound healing, and may be essential in the early stages of initiation of the limb regeneration cascade.

Mouse limb bud cells respond to retinoic acid in vitro with reduced growth.

Retinoic acid (RA) has dramatic effects on the pattern of developing and regenerating vertebrate limbs. These effects are considered to result from RA-induced changes in the positional identity of limb cells, and involve the formation of extra structures. Whether the growth required to form the supernumerary parts of the pattern is a primary effect of RA treatment or a secondary effect that follows after a change in positional identity is not at present known. In this paper we have investigated the effects of RA treatment on the growth of cells from anterior and posterior halves of mouse limb buds in vitro. We observed that under our culture conditions, limb bud cells treated with 1 nM to 1 microM RA (0.3 ng/ml to 300 ng/ml) continue to grow but do so at a significantly slower rate than control cultures. There is a maximum inhibition of growth (50% of controls) between 10 nM and 100 nM RA, which corresponds to the measured range of concentrations of RA in vivo. Our observation of a significant decrease in growth rate over a wide range of RA concentrations is consistent with comparable reports of growth inhibition for a large number of other cell types in vitro as well as with the observation that exogenous RA inhibits blastemal growth in amphibians during the period of exposure to RA. We propose that the effects of RA on growth, either enhancement in vivo or reduction in vitro, can be seen as consequences of the ability of RA to alter positional identity.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of amitriptyline on pain intensity and perception of stress in bruxers.

PURPOSE: The purpose of this clinical pilot study was to evaluate the effect of a tricyclic antidepressant, amitriptyline, on pain-intensity level and level of stress in bruxers. MATERIALS AND METHODS: In a randomized, double-blind, crossover experimental design, 10 subjects received active (amitriptyline 25 mg/night) and inactive (placebo 25 mg/night) medication, over a period of 4 weeks. RESULTS: The administration of amitriptyline for 4 weeks did not significantly (p >.05) reduce pain intensity. However, it significantly (p <.05) reduced the level of stress perception. CONCLUSION: The results of this limited study do not support the administration of small doses of amitriptyline over a period of 4 weeks for the management of pain resulting from sleep bruxism. However, the results support the administration of small doses of amitriptyline for the management of the perception of stress levels associated with sleep bruxism.