Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70.

At least eight inherited human neurodegenerative diseases are caused by expansion of a polyglutamine domain within the respective proteins. This confers dominant toxicity on the proteins, leading to dysfunction and loss of neurons. Expanded polyglutamine proteins form aggregates, including nuclear inclusions (NI), within neurons, possibly due to misfolding of the proteins. NI are ubiquitinated and sequester molecular chaperone proteins and proteasome components, suggesting that disease pathogenesis includes activation of cellular stress pathways to help refold, disaggregate or degrade the mutant disease proteins. Overexpression of specific chaperone proteins reduces polyglutamine aggregation in transfected cells, but whether this alters toxicity is unknown. Using a Drosophila melanogaster model of polyglutamine disease, we show that directed expression of the molecular chaperone HSP70 suppresses polyglutamine-induced neurodegeneration in vivo. Suppression by HSP70 occurred without a visible effect on NI formation, indicating that polyglutamine toxicity can be dissociated from formation of large aggregates. Our studies indicate that HSP70 or related molecular chaperones may provide a means of treating these and other neurodegenerative diseases associated with abnormal protein conformation and toxicity.

Bone morphogenetic proteins and Smad expression in ovine tendon-bone healing.

PURPOSE: Bone morphogenetic proteins (BMPs) are being developed to improve tendon-bone healing. To do this, it is essential to understand the endogenous expression of BMPs and their downstream signal transduction factors, Smads, during tendon-bone healing. METHODS: An extra-articular patellar tendon-bone healing ovine model was set up, and histologic evaluation of the healing progress at the tendon-bone interface at 1, 2, 3, and 6 weeks was performed. Immunohistochemical staining of BMP-2, BMP-7, Smad1, Smad4, and Smad5 was carried out in all sections. RESULTS: The model revealed formation of a loose granuloma tissue layer between the tendon and bone at 1 week, remodeling starting at 2 weeks, and Sharpey-like collagen fiber formation at 3 and 6 weeks. All detected factors were elevated at the tendon-bone interface during healing, and the expression peaked at 2 to 3 weeks. The cells involved were osteoblastic-like cells, osteoclastic-like cells, mesenchymal cells, and fibroblasts. BMP-7 staining was mainly at the interface close to the bony side, whereas BMP-2 expression shifted to the tendon side at 6 weeks. The expression pattern of Smad1 and Smad5 was similar to that of BMP-7. Smad1 was also found to be expressed in osteoclastic-like cells at 1 and 2 weeks. Smad4 expression was the highest among all of the factors at all time points. CONCLUSIONS: The data suggest that endogenous BMP-2 and BMP-7 participate in tendon-bone healing and their functions involve their downstream signal transduction mediators, Smad1, Smad4, and Smad5. CLINICAL RELEVANCE: The temporal expression of BMPs should be considered when setting up therapeutic strategies using BMPs.

Hypomorphic mutations in the larval photokinesis A (lphA) gene have stage-specific effects on visual system function in Drosophila melanogaster.

Of the many genes that are expressed in the visual system of Drosophila melanogaster adults, some affect larval vision. However, with the exception of one X-linked mutation, no genes that have larval-specific effects on visual system structure or function have previously been reported. We describe the isolation and characterization of two mutant alleles that define the larval photokinesis A (lphA) gene, one allele of which is associated with a P-element insertion at cytogenetic locus 8E1-10. Larvae that express lphA mutations are, like normal animals, negatively photokinetic, but they are less responsive to white light than lphA + controls. Larvae that are heterozygous in trans for a mutant lphA allele and a deficiency that uncovers the lphA locus are blind, which indicates that the mutant allele is hypomorphic. lphA larvae respond normally to odorants and taste stimuli. Moreover, the lphA mutations do not affect adult flies' fast phototaxis or visually driven aspects of male sexual behavior, and electroretinograms recorded from the compound eyes of lphA/deficiency heterozygotes and lphA1/lphA2 females are normal. These observations suggest that the lphA gene affects a larval-specific aspect of visual system function.

Dental fluorosis: variability among different inbred mouse strains.

Concurrent with the decline in dental caries has been an increase in the prevalence of dental fluorosis, a side-effect of exposure to greater than optimal levels of fluoride during amelogenesis. The mechanisms that underlie the pathogenesis of dental fluorosis are not known. We hypothesize that genetic determinants influence an individual's susceptibility or resistance to develop dental fluorosis. We tested this hypothesis using a mouse model system (continuous eruption of the incisors) where genotype, age, gender, food, housing, and drinking water fluoride level can be rigorously controlled. Examination of 12 inbred strains of mice showed differences in dental fluorosis susceptibility/resistance. The A/J mouse strain is highly susceptible, with a rapid onset and severe development of dental fluorosis compared with that in the other strains tested, whereas the 129P3/J mouse strain is least affected, with minimal dental fluorosis. These observations support the contribution of a genetic component in the pathogenesis of dental fluorosis.

Effect of aging on animal response to chronic fluoride exposure.

This study was conducted to test the hypothesis that physiological changes which occur during aging increase the biological impact of fluoride and reduce the threshold of safe fluoride exposure. Four groups of rats were fed a low-fluoride diet (< 1.2 ppm) ad libitum and received 0, 5, 15, or 50 ppm fluoride in their drinking water. Animals were killed after three, six, 12, or 18 months of treatment. Blood and urine were monitored for biochemical markers of tissue function, and plasma, urine, feces, and representative tissues were analyzed for fluoride. In addition, bone marrow cells from animals killed after 18 months of treatment were examined for frequency of sister chromatid exchange (SCE), a marker of genetic damage. Study results indicated that, within treatment groups, fluoride intake, excretion, and retention did not change significantly between three and 18 months. Fluoride concentration in soft tissues did not change with treatment duration in the fluoride-treated animals. Mineralized tissue fluoride concentration and the total fluoride in the carcasses increased continually as the animals aged. In spite of significant, dose-related differences in tissue fluoride levels which occurred in all age groups in this study, there were no indications that increased fluoride in the tissues caused any adverse physiological or genotoxic effects. None of the monitored clinical "wellness" markers of tissue integrity and function was altered by fluoride in a clinically significant manner. Therefore, there was no evidence from this study that aging reduces the threshold of safe chronic fluoride exposure.

Absence of detrimental effects of fluoride exposure in diabetic rats.

This study is part of a comprehensive programme to investigate fluoride toxicity and the hypothesis that fluoride ingested by "medically compromised' animals will result in altered physiological function. Its objectives were to monitor fluoride retention, tissue fluoride concentrations and genetic variables in diabetic and control rats chronically exposed to fluoride, and to determine whether or not adverse effects occurred. Male, Zucker fatty diabetic rats and Zucker age-matched lean controls were fed a low-fluoride diet ( < 1.2 parts/10(6) F-) ad libitum and received 0, 5, 15 or 50 parts/10(6) fluoride in their drinking water for 3 or 6 months. Fluoride metabolic balance was determined for 4 days before the end of each study phase. Plasma and urine were analysed for biochemical markers of tissue function, and plasma, urine, faeces and tissues were analysed for fluoride. Bone marrow cells from animals killed after 6 months of treatment were examined for frequency of sister chromatid exchange, a marker of genetic damage. The diabetic rats consumed, excreted and retained significantly greater amounts of fluoride than the controls (p < 0.05). There were dose-related increases in fluoride excretion, retention and tissue concentrations in both classes of animals, which were significantly greater in the diabetic rats. In spite of greater amounts of fluoride in the tissues of diabetic animals, there was no evidence, under these experimental conditions, that any of the fluoride exposures tested caused measurable adverse effects on the physiological, biochemical or genetic variables that were monitored.

Effect of sodium hexametaphosphate on dental calculus formation in dogs.

A series of studies was conducted to identify a practical measure for preventing dental calculus formation in dogs. The studies involved a colony of 27 Beagles that received an initial dental prophylaxis. The dogs were then stratified on the basis of their normal rate of calculus formation and randomly assigned to parallel groups within each strata. During 4-week test periods, a variety of experimental regimens were instituted, followed by clinical assessments of calculus. Major observations were that a crystal growth inhibitor, soluble pyrophosphate, incorporated into a dry dog food modestly reduced calculus formation when used at high concentration; anticalculus effects attributable to this agent were significant (P < 0.05) only when it was used as a surface coating; the coating of dry dog chow or plain biscuits with a calcium sequestrant, sodium hexametaphosphate (HMP), provided the greatest benefit and resulted in significant (P < 0.05) reductions in calculus formation of about 60 to 80%, depending on the dosage regimen; and the feeding of a single daily snack of 2 HMP-coated plain biscuits (0.6% HMP) decreased calculus formation by nearly 80%. We concluded that the coating of dry dog chow or plain dog biscuits with HMP is an effective means of reducing calculus formation in dogs.

Caries-preventive effects of sodium and amine fluoride dentifrices.

PURPOSE: To determine the comparative effects of sodium (NaF) and amine fluoride dentifrices, as well as the effect of different silica abrasives used in NaF dentifrices, on caries formation in rats. MATERIALS AND METHODS: Harlan Sprague Dawley mixed sex, weanling, littermate rats were fed a cariogenic diet and inoculated with S. sobrinus strain 6715 to induce caries formation. The dentifrices evaluated contained 0.31% NaF with precipitated silica I as the abrasive system; 0.31% NaF with sodium bicarbonate and hydrated silica; placebo; amine fluoride (0.14%) containing silica (Elmex); 0.31% NaF with precipitated silica II (Sensodyne F); and 0.31% NaF with hydrated silica. Dentifrice slurries were administered 7 days per week for 3 weeks at which time the animals were euthanized and the hemijaws removed, coded and stained with a murexide solution. The recovery of S. sobrinus after the 3-week study clearly showed that all of the six groups were infected with S. sobrinus. The Keyes scoring method was then used to assess the caries formation within the six groups. RESULTS: Caries scores showed that all five experimental fluoride dentifrices were equally efficacious statistically in reducing caries formation when compared to the placebo with reductions ranging from 26 to 43%.

Effect of clindamycin hydrochloride on oral malodor, plaque, calculus, and gingivitis in dogs with periodontitis.

Periodontal disease is considered the most common disease of dogs and cats. The clinical sign most frequently reported by clients is oral malodor. Clindamycin hydrochloride has been used for several years for the treatment of periodontal disease in both cats and dogs. This study was designed to assess the effect of clindamycin HCl when used in two different post-prophylaxis treatment regimens on oral malodor and periodontal disease in a controlled clinical trial. This study demonstrated that a 5-day postprophylaxis regimen was significantly effective in reducing oral malodor compared with a dental prophylaxis alone. Clindamycin HCl significantly reduced oral malodor from the animals' baseline levels through 42 days. In addition, although no effect was observed on periodontal pocket depth, this treatment regimen also resulted in significant reductions in dental plaque, dental calculus, and gingival bleeding.

Preventing dental calculus formation in lemurs (Lemur catta, Eulemur fulvus collaris) and baboons (Papio cynocephalus).

The prevention of calculus accumulation in exotic animals is a relatively unexplored topic. A 6-mo study in ring-tailed lemurs (Lemur catta) and collared lemurs (Eulemur fulvus collaris) and two studies in baboons (Papio cynocephalus) (7.5 wks and 6.5 mo) tested the benefits of a primate diet coated with 0.6% sodium hexametaphosphate (HMP) in controlling calculus in these species using a sequential crossover design. The control regimen was an identical, but non-HMP-coated, dry primate chow. At study initiation, the primates were given a thorough dental prophylaxis and provided with the control diet or experimental diet. At the conclusion of the test period, the animals were anesthetized and examined for clinical calculus independently by two examiners. The animals were then given another dental prophylaxis, provided the alternate ration, and the foregoing procedures were repeated. When the animals were provided the HMP-coated diet, significant reductions in calculus formation of 48-62% were observed in the lemurs and the baboons. No clinically significant changes were observed in body weights or in blood chemistry values as a result of ingestion of the HMP-coated regimen.

Midterm results using a medial pivot total knee replacement compared with the Australian National Joint Replacement Registry data.

BACKGROUND: In order to emulate normal knee kinematics more closely, and thereby potentially improve wear characteristics and implant longevity, the medial pivot-type knee replacement geometry was designed. In the current study the outcome of 50 consecutive knee replacements using a medial pivot-type knee replacement was compared with the results in the Australian Orthopaedic Association National Joint Replacement Registry. METHODS: Pre- and post-operatively at follow-up evaluation consisted of the Knee Society score system and the Western Ontario and McMaster Universities Arthritis Index Score. Patient satisfaction was documented using 5-point Likert-type scales. Standard radiographs were used to assess signs of radiographic failure. Revisions were subcategorized into major total, major partial and minor. Patient records were cross-referenced against the Australian Orthopaedic Associations National Joint Replacement Registry's and the outcome compared with the registry's subset of data on the medial pivot knee used. RESULTS: According to the patients' Knee Society score system and Western Ontario and McMaster Universities Arthritis Index scores, there was good pain relief and functional improvement; none of the implants showed radiographic signs of failure. There was one minor revision. There was no statistically significant difference in revision rate compared with the registry results. DISCUSSION: The medial pivot knee-type implant in this series provided pain relief, functional improvement and a revision rate, similar to what is reported in the literature after a longer follow-up period, which is reassuring for those who use this type of implant on a day-to-day basis.

Spectral sensitivity of wild-type and mutant Drosophila melanogaster larvae.

Wild-type (Canton-S) Drosophila melanogaster larvae are generally repelled by white light. Mutant larval photokinesis A (lphA) larvae are less strongly repelled than controls. Mutant Larval photokinesis B (LphB) larvae are unresponsive to light, as are larvae from LI2, an isofemale line whose progenitors were recently derived from a natural population. To characterize the behavior of larvae from the mutant stocks and the isofemale line more precisely, we determined the range of wavelengths that repel wild-type (Canton-S) D. melanogaster larvae and identified wavelengths to which larvae are most sensitive. In comparison to adult flies, Canton-S larvae are much less sensitive to white light and respond to a narrower range of wavelengths. The wavelengths to which Canton-S larvae are maximally sensitive are 500 nm (green), 420 nm (indigo), and 380 nm (ultraviolet). Mutant lphA larvae respond abnormally to green and indigo light but are as strongly repelled by ultraviolet light as controls. In contrast, mutant LphB larvae and larvae from the LI2 isofemale line are unresponsive to green, indigo, or ultraviolet light. Thus, lphA larvae have a wavelength-specific defect, while LphB and LI2 larvae are generally unresponsive to wavelengths that repel wild-type larvae.

The Drosophila eyes absent gene directs ectopic eye formation in a pathway conserved between flies and vertebrates.

The fly eyes absent (eya) gene which is essential for compound eye development in Drosophila, was shown to be functionally replaceable in eye development by a vertebrate Eya homolog. The relationship between eya and that of the eyeless gene, a Pax-6 homolog, critical for eye formation in both flies and man, was defined: eya was found to be essential for eye formation by eyeless. Moreover, eya could itself direct ectopic eye formation, indicating that eya has the capacity to function as a master control gene for eye formation. Finally, we show that eya and eyeless together were more effective in eye formation than either gene alone. These data indicate conservation of the pathway of eya function between flies and vertebrates; they suggest a model whereby eya/Eya gene function is essential for eye formation by eyeless/Pax-6, and that eya/Eya can in turn mediate, via a regulatory loop, the activity of eyeless/Pax-6 in eye formation.

Animal caries models for evaluating fluoride dentifrices.

To determine the ability of animal caries models to evaluate the cariostatic potential of fluoride dentifrices, we examined four different coronal caries models. These currently used rat caries models were: Francis' hypomineralized area model, Gaffar's CARA rat model, the Connecticut model, and the Indiana model. Available data indicated that all of these models were capable of detecting significant cariostatic benefits from clinically proven fluoride dentifrices. For the most part, these animal models were also able to demonstrate a response to increasing concentrations of fluoride, particularly if the fluoride was provided in an ionic rather than a complexed form. Data were also presented which indicated the potential for animal caries models to be used to predict the clinical benefits of non-fluoride systems as well as the utility of fluoride systems for the prevention of root-surface caries. It is concluded that animal caries models play a significant role in the development and evaluation of fluoride and other systems for the clinical prevention of dental caries.

Mechanisms of chaperone suppression of polyglutamine disease: selectivity, synergy and modulation of protein solubility in Drosophila.

At least eight dominant human neurodegenerative diseases are due to the expansion of a polyglutamine within the disease proteins. This confers toxicity on the proteins and is associated with nuclear inclusion formation. Recent findings indicate that molecular chaperones can modulate polyglutamine pathogenesis, but the basis of polyglutamine toxicity and the mechanism by which chaperones suppress neurodegeneration remains unknown. In a Drosophila: disease model, we demonstrate that chaperones show substrate specificity for polyglutamine protein, as well as synergy in suppression of neurotoxicity. Our analysis also reveals that chaperones alter the solubility properties of the protein, indicating that chaperone modulation of neurodegeneration in vivo is associated with altered biochemical properties of the mutant polyglutamine protein. These findings have implications for these and other human neurodegenerative diseases associated with abnormal protein aggregation.

Interstrain variability of larval photokinesis in Drosophila melanogaster.

Larvae from seven laboratory strains and eight isofemale lines of Drosophila melanogaster differ significantly with regard to their responses to light in a photokinesis assay in which the larvae are tested en masse. Larvae from the CA-2 laboratory stock fail to disperse on assay plates, although observations of individual CA-2 larvae suggest that the larvae are repelled by light. Larvae from all of the other laboratory stocks and all of the isofemale lines (except LI2 and NC5) avoid light in the photokinesis assay. Larvae from some stocks are much more strongly repelled by light than larvae from other stocks. LI2 larvae are unresponsive to light in most replicates of the photokinesis assay, while NC5 larvae are consistently unresponsive to light. Observations of F1 heterozygotes suggest that the allele(s) that affects the vision of LI2 and NC5 larvae has net effects on the animals' behavior that are partially dominant and recessive, respectively.

Expanded polyglutamine protein forms nuclear inclusions and causes neural degeneration in Drosophila.

Spinocerebellar ataxia type 3 (SCA3/MJD) is one of at least eight human neurodegenerative diseases caused by glutamine-repeat expansion. We have recreated glutamine-repeat disease in Drosophila using a segment of the SCA3/MJD protein. Targeted expression of the protein with an expanded polyglutamine repeat led to nuclear inclusion (NI) formation and late-onset cell degeneration. Differential sensitivity to the mutant transgene was observed among different cell types, with neurons being particularly susceptible; NI formation alone was not sufficient for degeneration. The viral antiapoptotic gene P35 mitigated polyglutamine-induced degeneration in vivo. Our results demonstrate that cellular mechanisms of human glutamine-repeat disease are conserved in invertebrates. This fly model will aid in identifying additional factors that modulate neurodegeneration.