An ultrastructural study of dentinogenesis and amelogenesis in rat molar tooth germs cultured in vitro

Summary Molar tooth germs from three-day-old rats were cultured successfully for fourteen days, permitting the study of the development in vitro of both extracellular matrix and cellular elements such as odontoblasts and ameloblasts. The ultrastructure of the cultured tooth germs was compared with the ultrastructure of tooth germs in vivo at a comparable developmental stage. Progenitor cells of odontoblasts and ameloblasts were found to differentiate in vitro. Odontoblasts seemed to contain more lysosome-like bodies and fewer secretory granules than in vivo. They formed normally mineralizing dentine or a thick layer of dense, unmineralized predentine with incidentally some amorphous, extracellular material. Enamel was exclusively present opposite well developed dentine. It was often hyperor hypomineralized and enamel rods were not as regularly shaped as in vivo. In places where no enamel formation had taken place, large amounts of amorphous extracellular material were sometimes seen. From these observations it can be concluded that cellular development in cultured tooth germs appeared more or less normal, but extracellular matrix formation and mineralization were sometimes disturbed.     

A novel technique for rapid determination of energy consumption in platelets. Demonstration of different energy consumption associated with three secretory responses

A novel method has been developed for rapid and quantitative determination of the rate of energy consumption in platelets. In platelets suspended in a cyanide-containing medium. ATP resynthesis is abruptly blocked by addition of 2-deoxyglucose and D-glucono-1,5-lactone. We demonstrate that the subsequent changes in the levels of cytoplasmic ATP and ADP reflect the velocity of energy consumption in the platelets immediately before addition of the inhibitors. Despite the arrest in ATP resynthesis the platelets remain responsive to stimulation by thrombin (5 units x ml-1) which triggers the secretion of the contents of dense, alpha- and acid hydrolase granules. Unstimulated platelets were found to consume about 3.5 and 0.5 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at 37 degrees C and 15 degrees C, respectively; the thrombin-treated platelets consumed respectively 16 and 2 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at these temperatures. When the velocity of energy consumption was varied by (a) changing the temperature and (b) preincubation with glyco(geno)lytic inhibitors, it was found to be linearly related to the initial rate of secretion from the three types of granules. The precise nature of this relationship differed between the three types of secretion responses and indicated an increasing requirement for metabolic energy for secretion from the three types of granules in the order: dense granule less than alpha-granule less than acid hydrolase granule. The results obtained with changes in temperature were superimposable on those obtained with the glyco(geno)lytic inhibitors for dense granule secretion and alpha-granule secretion, suggesting an apparent coupling between energy consumption and the rate of these secretion responses. The rate of secretion of acid hydrolase was always higher when energy consumption was varied by temperature changes than when glyco(geno)lytic inhibitors were used, probably as a result of metabolic changes prior to induction of secretion. On the basis of these experiments, we calculated an incremental energy consumption during complete secretion of dense, alpha- and acid hydrolase granule contents of 2.5, 4.2 and 6.7 mumol of ATP equivalents x (10(11) platelets)-1, respectively.     

Ultrastructural localization of dentine phosphoprotein in rat tooth germs by immunogold staining

Summary Dentine phosphoprotein (DPP) was localized on thin frozen sections of fixed rat tooth germs by indirect immunogold staining. Antisera were directed against DPP and against glutaraldehyde-treated DPP and were characterized by immuno-electroblotting. In odontoblasts, DPP was found to be localized in the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus and in Golgi-associated vesicles. Odontoblastic processes were moderately positive for DPP and dentine was intensely labeled on frozen sections of unfixed tissue. Predentine showed a slight immunoreactivity. These results indicate the synthesis of DPP in the RER, its accumulation in the Golgi apparatus and its vesicular transport and secretion via the odontoblastic processes into dentine. The close association of the gold particles with the dentinal collagen fibres makes a role of DPP in linking mineral to collagen conceivable. Matrix vesicles were negative for DPP, suggesting that the protein is not present at the sites of matrix vesicleassociated nucleation.     

Application of an optimized marker amplification protocol in immunohistochemistry — a valuable tool for visualizing antigenic sites of low signal strength or sparse distribution

Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue.     

EFFECT OF VITAMIN E SUPPLEMENTATION AND PARTIAL SUBSTITUTION OF POLY- WITH MONO-UNSATURATED FATTY ACIDS IN PIG DIETS ON MUSCLE,AND MICROSOME EXTRACT a-TOCOPHEROL CONCENTRATION AND LIPID OXIDATION

The experiment was organized in a 3×2 factorial arrangement with three dietary fat blends and a basal (20 mg kg^?1 diet) or supplemented (220 mg kg^?1) level of α-tocopheryl acetate. Dietary vitamin E and monounsaturated to polyunsaturated fatty acid ratio (dietary MUFA/PUFA) affected muscle α-tocopherol concentration (α-tocopherol [log μg g^?1]=0.18 (±0.105)+0.0034 (±0.0003)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.39 (±0.122)·dietary MUFA/PUFA (P<0.0036)). An interaction between dietary α-tocopherol and dietary MUFA/PUFA exists for microsome α-tocopherol concentration (α-tocopherol [log μg g^?1]=1.14 (±0.169) (P<0.0001)+0.0056 (±0.00099)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.54 (±0.206)·dietary MUFA/PUFA (P<0.0131)?0.0033 (±0.0011)·dietary α-tocopherol [mg kg^?1)]×dietary MUFA/PUFA (P<0.0067)), and hexanal concentration in meat (hexanal [ng·g^?1]=14807.9 (±1489.8)?28.8 (±10.6) dietary α-tocopherol [mg·kg^?1] (P<0.01)?8436.6 (±1701.6)·dietary MUFA/PUFA (P<0.001)+24.0 (±11.22)·dietary α-tocopherol·dietary MUFA/PUFA (P<0.0416)). It is concluded that partial substitution of dietary PUFA with MUFA lead to an increase in the concentration of α-tocopherol in muscle and microsome extracts. An interaction between dietary α-tocopherol and fatty acids exists, in which at low level of dietary vitamin E inclusion, a low MUFA/PUFA ratio leads to a reduction in the concentration of α-tocopherol in microsome extracts and a concentration of hexanal in meat above the expected values.     

Antisense-Oligonucleotide Mediated Exon Skipping in Activin-Receptor-Like Kinase 2: Inhibiting the Receptor That Is Overactive in Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare heritable disease characterized by progressive heterotopic ossification of connective tissues, for which there is presently no definite treatment. A recurrent activating mutation (c.617G→A; R206H) of activin receptor-like kinase 2 (ACVR1/ALK2), a BMP type I receptor, has been shown as the main cause of FOP. This mutation constitutively activates the BMP signaling pathway and initiates the formation of heterotopic bone. In this study, we have designed antisense oligonucleotides (AONs) to knockdown mouse ALK2 expression by means of exon skipping. The ALK2 AON could induce exon skipping in cells, which was accompanied by decreased ALK2 mRNA levels and impaired BMP signaling. In addition, the ALK2 AON potentiated muscle differentiation and repressed BMP6-induced osteoblast differentiation. Our results therefore provide a potential therapeutic approach for the treatment of FOP disease by reducing the excessive ALK2 activity in FOP patients.     

Quirks of dye nomenclature. 5. Rhodamines

Rhodamines were first produced in the late 19^th century, when they constituted a new class of synthetic dyes. These compounds since have been used to color many things including cosmetics, inks, textiles, and in some countries, food products. Certain rhodamine dyes also have been used to stain biological specimens and currently are widely used as fluorescent probes for mitochondria in living cells. The early history and current biological applications are sketched briefly and an account of the ambiguities, complications and confusions concerning dye identification and nomenclature are discussed.     

Quirks of dye nomenclature. 6. Malachite green

Malachite green was discovered independently by two researchers in Germany in the 19^th century and found immediate employment as a dye and a pigment. Subsequently, other uses, such as staining biological specimens, emerged. A much later application was the control of fungal and protozoan infections in fish, for which the dye remains popular, although illegal in many countries owing to a variety of toxicity problems. In solution, malachite green can exist as five different species depending on the pH. The location of the positive charge of the colored cation on a carbon atom or a nitrogen atom is still debated. The original names of this dye, and their origins, are briefly surveyed.     

Upregulation of Neuronal Nitric Oxide Synthase in in Vitro Stellate Astrocytes and in Vivo Reactive Astrocytes After Electrically Induced Status Epilepticus

Neuronal nitric oxide synthase (nNOS) is a constitutively expressed and calcium-dependent enzyme. Despite predominantly expressed in neurons, nNOS has been also found in astrocytes, although at lower expression levels. We have studied the regulation of nNOS expression in cultured rat astrocytes from cortex and spinal cord by Western blotting and immunocytochemistry. nNOS was not detectable in cultured astrocytes grown in serum-containing medium (SCM), but was highly expressed after serum deprivation. Accordingly, calcium-dependent NOS activity and both intracellular nitrite levels and nitrotyrosine immunoreactivity after glutamate stimulation were higher in serum-deprived astrocytes than in cells grown in SCM. Serum deprivation induced a modification of astrocytes morphology, from flat to stellate. nNOS upregulation was also observed in reactive astrocytes of rat hippocampi after electrically induced status epilepticus, as demonstrated by double-labeling experiments. Thus, nNOS upregulation occurs in both in vitro stellate and in vivo reactive astrocytes, suggesting a possible involvement of glial nNOS in neurological diseases characterized by reactive gliosis.