Reversible and Irreversible Effects of Temperature on Amelogenesis of Hamster Tooth Germs In Vitro

Hamster first hamster molar tooth germs in early secretory stage of amelogenesis were cultured for one day in vitro at 6°C, 22°C, 37°C or 45°C in the presence of 3H-proline, 45Ca and 32P-orthophosphate. Other explants were cultured without these labels and after culture examined by histology. The highest temperature tested was lethal to the explants, decreased total dry weight and rapidly increased total uptake of the radio-labelled mineral ions, probably merely due to physicochemical modification of the existing preculture minerals. Optimal synthesis and secretion of amelogenins were measured at physiological temperature (37°C). Effects of exposure to both temperatures below the physiological value were virtually reversible when explants were grown at physiological temperature (37°C) for another day. However, amelogenin secretion during this recovery period did not reach values as high as those found for the first day in explants initially grown at physiological temperature during the first day. We concluded from the four temperatures examined that the optimal temperature for enamel matrix deposition in vitro was 37°C. At this temperature enamel biosynthesis and its secretion are high. Lowering the temperature slows down the metabolism without any apparent harmful effect. Normal development of the tooth explants in vitro resumes when the culture temperature is restored to physiological levels (37°C). For temporary storage of tooth germ explants prior to any reimplantation, we therefore recommend a temperature of 6°C

Micro-PIXE (Proton-Induced X-Ray Emission) Study of the Effects of Fluoride on Mineral Distribution Patterns in Enamel and Dentin in the Developing Hamster Tooth Germ

Micro-PIXE (proton-induced X-ray emission) analysis was performed on unfixed and anhydrously prepared sections from developing enamel and dentin from hamsters injected with a single dose of 20 mg NaF /kg body weight. Fluoride, apart from inducing the formation of the characteristic paired response in the enamel (i.e., a hyper- followed by a hypomineralized band in the secretory enamel), also induces the formation of sub-ameloblastic cystic lesions under the transitional and early secretory enamel accompanied by relatively intense hypermineralization of the underlying cystic enamel surface. These cystic lesions, however, were only found to be associated with certain isolated populations of these cells. In addition, these lesions were restricted to the smooth surfaces of the tooth germ only. Cystic lesions such as those seen under the transitional and early secretory ameloblasts were not observed under the fully secretory or maturation stage ameloblasts. Why fluoride induces the formation of cystic lesions in some ameloblast populations while other cells in the same stage of development apparently remain unaffected, is a matter which needs further investigation

The effect of fluoride on enamel and dentin formation in the uremic rat incisor

Renal impairment in children is associated with tooth defects that include enamel pitting and hypoplasia. However, the specific effects of uremia on tooth formation are not known. In this study, we used rat mandibular incisors, which continuously erupt and contain all stages of tooth formation, to characterize the effects of uremia on tooth formation. We also tested the hypothesis that uremia aggravates the fluoride (F)-induced changes in developing teeth. Rats were subjected to a two-stage 5/6 nephrectomy or sham operation and then exposed to 0 (control) or 50 ppm NaF in drinking water for 14 days. The effects of these treatments on food intake, body growth rate, and biochemical serum parameters for renal function and calcium metabolism were monitored. Nephrectomy reduced food intake and weight gain. Intake of F by nephrectomized rats increased plasma F levels twofold and further decreased food intake and body weight gain. Uremia affected formation of dentin and enamel and was more extensive than the effect of F alone. Uremia also significantly increased predentin width and induced deposition of large amounts of osteodentin-like matrix-containing cells in the pulp chamber. In enamel formation, the cells most sensitive to uremia were the transitional-stage ameloblasts. These data demonstrate that intake of F by rats with reduced renal function impairs F clearance from the plasma and aggravates the already negative effects of uremia on incisor tooth development

Multiple Trypanosoma infections are common amongst Glossina species in the new farming areas of Rufiji district, Tanzania

<p>Abstract</p> <p>Background</p> <p>Tsetse flies and trypanosomiasis are among several factors that constrain livestock development in Tanzania. Over the years Rufiji District was excluded from livestock production owing to tsetse fly infestation, however, a few years ago there was an influx of livestock following evictions aimed at conserving the Usangu wetlands.</p> <p>Methods</p> <p>A study was conducted to determine the efficiency of available traps for catching tsetse flies, <it>Glossina </it>species infesting the area, their infection rates and <it>Trypanosoma </it>species circulating in the area. Trapping was conducted during the semi dry season for a total of 30 days (ten days each month) during the onset of the dry season of May - July 2009. Harvested flies after every 24 hours were dissected and examined under a light microscope for trypanosome infections and whole fly DNA was extracted from 82 flies and analyzed for trypanosomes by polymerase chain reaction (PCR) using different sets of primers.</p> <p>Results</p> <p>The proportions of total tsetse catches per trap were in the following decreasing order S3 (33%), H-Trap (27%), Pyramidal (19%), sticky panel (11%) and biconical trap (10%). Of the 1200 trapped flies, 75.6% were identified as <it>Glossina pallidipes</it>, 11.7% <it>as G. brevipalpis</it>, 9.6% as <it>G. austeni </it>and 3.0% <it>G. morsitans morsitans</it>. Dissections revealed the overall infection rate of 6.6% (13/197). Whole DNA was extracted from 82 tsetse flies and the prevalence of trypanosomes circulating in the area in descending order was 92.7% (76/82) for <it>T. simiae</it>; 70.7% (58/82) for <it>T. brucei </it>types; 48.8% (40/82) for the <it>T. vivax </it>types and 32.9% (27/82) for the <it>T. congolense </it>types as determined by PCR. All trypanosome types were found in all tsetse species analysed except for the <it>T. congolense </it>types, which were absent in <it>G. m. morsitans</it>. None of the <it>T. brucei </it>positive samples contained human infective trypanosomes by SRA - PCR test</p> <p>Conclusion</p> <p>All tsetse species found in Rufiji are biologically important in the transmission of animal trypanosomiasis and the absence of <it>T. congolense </it>in <it>G. m. morsitans </it>could be a matter of chance only. Therefore, plans for control should consider all tsetse species.</p

Reduced Protein Expression of the Na+/Ca2++K+-Exchanger (SLC24A4) in Apical Plasma Membranes of Maturation Ameloblasts of Fluorotic Mice

Exposure of forming enamel to fluoride results into formation of hypomineralized enamel. We tested whether enamel hypomineralization was caused by lower expression of the NCKX4/SLC24A4 Ca2+-transporter by ameloblasts. Three commercial antibodies against NCKX4 were tested on enamel organs of wild-type and Nckx4-null mice, one of which (a mouse monoclonal) was specific. This antibody gave a prominent staining of the apical plasma membranes of maturation ameloblasts, starting at early maturation. The layer of immuno-positive ameloblasts contained narrow gaps without immunostaining or with reduced staining. In fluorotic mouse incisors, the quantity of NCKX4 protein in ameloblasts as assessed by western blotting was not different from that in non-fluorotic ameloblasts. However, immunostaining of the apical plasma membranes of fluorotic ameloblasts was strongly reduced or absent suggesting that trafficking of NCKX4 to the apical membrane was strongly reduced. Exposure to fluoride may reduce NCKX4-mediated transport of Ca2+ by maturation stage ameloblasts which delays ameloblast modulation and reduces enamel mineralization

Prevalence of Malaria Parasitemia and Purchase of Artemisinin-Based Combination Therapies (ACTs) among Drug Shop Clients in Two Regions in Tanzania with ACT Subsidies.

Throughout Africa, many people seek care for malaria in private-sector drug shops where diagnostic testing is often unavailable. Recently, subsidized artemisinin-based combination therapies (ACTs), a first-line medication for uncomplicated malaria, were made available in these drug shops in Tanzania. This study assessed the prevalence of malaria among and purchase of ACTs by drug shop clients in the setting of a national ACT subsidy program and sub-national drug shop accreditation program. A cross-sectional survey of drug shop clients was performed in two regions in Tanzania, one with a government drug shop accreditation program and one without, from March-May, 2012. Drug shops were randomly sampled from non-urban districts. Shop attendants were interviewed about their education, training, and accreditation status. Clients were interviewed about their symptoms and medication purchases, then underwent a limited physical examination and laboratory testing for malaria. Malaria prevalence and predictors of ACT purchase were assessed using univariate analysis and multiple logistic regression. Amongst 777 clients from 73 drug shops, the prevalence of laboratory-confirmed malaria was 12% (95% CI: 6-18%). Less than a third of clients with malaria had purchased ACTs, and less than a quarter of clients who purchased ACTs tested positive for malaria. Clients were more likely to have purchased ACTs if the participant was <5 years old (aOR: 6.6; 95% CI: 3.9-11.0) or the shop attendant had >5 years, experience (aOR: 2.8; 95% CI: 1.2-6.3). Having malaria was only a predictor of ACT purchase in the region with a drug shop accreditation program (aOR: 3.4; 95% CI: 1.5-7.4).\ud Malaria is common amongst persons presenting to drug shops with a complaint of fever. The low proportion of persons with malaria purchasing ACTs, and the high proportion of ACTs going to persons without malaria demonstrates a need to better target who receives ACTs in these drug shops

Mutations in the pH-Sensing G-protein-Coupled Receptor GPR68 Cause Amelogenesis Imperfecta

Amelogenesis is the process of dental enamel formation, leading to the deposition of the hardest tissue in the human body. This process requires the intricate regulation of ion transport and controlled changes to the developing enamel matrix pH. The means by which the enamel organ regulates pH during amelogenesis is largely unknown. We identified rare homozygous variants in GPR68 in three families with Amelogenesis Imperfecta, a genetically and phenotypically heterogeneous group of inherited conditions associated with abnormal enamel formation. Each of these homozygous variants (a large in-frame deletion, a frameshift deletion and a missense) were predicted to result in loss of function. GPR68 encodes a proton sensing G-protein-coupled receptor with sensitivity in the pH range that occurs in the developing enamel matrix during amelogenesis. Immunohistochemistry of rat mandibles confirmed localisation of GPR68 in the enamel organ at all stages of amelogenesis. Our data identify a role for GPR68 as a proton sensor that is required for proper enamel formation

Mutations in the Beta Propeller WDR72 Cause Autosomal-Recessive Hypomaturation Amelogenesis Imperfecta

Healthy dental enamel is the hardest and most highly mineralized human tissue. Though acellular, nonvital, and without capacity for turnover or repair, it can nevertheless last a lifetime. Amelogenesis imperfecta (AI) is a collective term for failure of normal enamel development, covering diverse clinical phenotypes that typically show Mendelian inheritance patterns. One subset, known as hypomaturation AI, is characterised by near-normal volumes of organic enamel matrix but with weak, creamy-brown opaque enamel that fails prematurely after tooth eruption. Mutations in genes critical to enamel matrix formation have been documented, but current understanding of other key events in enamel biomineralization is limited. We investigated autosomal-recessive hypomaturation AI in a consanguineous Pakistani family. A whole-genome SNP autozygosity screen identified a locus on chromosome 15q21.3. Sequencing candidate genes revealed a point mutation in the poorly characterized WDR72 gene. Screening of WDR72 in a panel of nine additional hypomaturation AI families revealed the same mutation in a second, apparently unrelated, Pakistani family and two further nonsense mutations in Omani families. Immunohistochemistry confirmed intracellular localization in maturation-stage ameloblasts. WDR72 function is unknown, but as a putative β propeller is expected to be a scaffold for protein-protein interactions. The nearest homolog, WDR7, is involved in vesicle mobilization and Ca2+-dependent exocytosis at synapses. Vesicle trafficking is important in maturation-stage ameloblasts with respect to secretion into immature enamel and removal of cleaved enamel matrix proteins via endocytosis. This raises the intriguing possibility that WDR72 is critical to ameloblast vesicle turnover during enamel maturation

Regulation of pH During Amelogenesis

During amelogenesis, extracellular matrix proteins interact with growing hydroxyapatite crystals to create one of the most architecturally complex biological tissues. The process of enamel formation is a unique biomineralizing system characterized first by an increase in crystallite length during the secretory phase of amelogenesis, followed by a vast increase in crystallite width and thickness in the later maturation phase when organic complexes are enzymatically removed. Crystal growth is modulated by changes in the pH of the enamel microenvironment that is critical for proper enamel biomineralization. Whereas the genetic bases for most abnormal enamel phenotypes (amelogenesis imperfecta) are generally associated with mutations to enamel matrix specific genes, mutations to genes involved in pH regulation may result in severely affected enamel structure, highlighting the importance of pH regulation for normal enamel development. This review summarizes the intra- and extracellular mechanisms employed by the enamel-forming cells, ameloblasts, to maintain pH homeostasis and, also, discusses the enamel phenotypes associated with disruptions to genes involved in pH regulation