Comparison of decalcifying agents and techniques for human dental tissues.

Teeth are among the hardest animal tissues, because they are composed of large amounts of inorganic compounds. Consequently, teeth are difficult to prepare for microscopic examination. Acids and chelating agents traditionally have been used to remove calcium ions. We compared decalcifying agents including strong acids, weak acids, chelating agents, techniques using electric current, agitation and heat. Freshly extracted teeth were fixed and decalcified using formic acid-formalin, formal-nitric acid, formalin-EDTA, Von Ebner's fluid and Perenyi's fluid. Three additional techniques including formic acid with agitation, formic acid with heat and formic acid with electric current also were evaluated. Decalcified teeth were evaluated histologically for tissue preservation and staining characteristics. Formic acid with gentle agitation produced the best decalcification overall based on time required for decalcification, ease of sectioning, hard and soft tissue staining and tissue preservation. Our findings support the use of agitation with formic acid decalcification, because it reduces significantly both the time required and the deleterious effects of prolonged immersion.

Enhanced phosphoinositide 3-kinase(p110α) activity prevents diabetes-induced cardiomyopathy and superoxide generation in a mouse model of diabetes.

AIMS/HYPOTHESIS: Diabetic cardiomyopathy is characterised by diastolic dysfunction, oxidative stress, fibrosis, apoptosis and pathological cardiomyocyte hypertrophy. Phosphoinositide 3-kinase (PI3K)(p110α) is a cardioprotective kinase, but its role in the diabetic heart is unknown. The aim of this study was to assess whether PI3K(p110α) plays a critical role in the induction of diabetic cardiomyopathy, and whether increasing PI3K(p110α) activity in the heart can prevent the development of cardiac dysfunction in a setting of diabetes. METHODS: Type 1 diabetes was induced with streptozotocin in adult male cardiac-specific transgenic mice with increased PI3K(p110α) activity (constitutively active PI3K [p110α], caPI3K] or decreased PI3K(p110α) activity (dominant-negative PI3K [p110α], dnPI3K) and non-transgenic (Ntg) mice for 12 weeks. Cardiac function, histological and molecular analyses were performed. RESULTS: Diabetic Ntg mice displayed diastolic dysfunction and increased cardiomyocyte size, expression of atrial and B-type natriuretic peptides (Anp, Bnp), fibrosis and apoptosis, as well as increased superoxide generation and increased protein kinase C ß2 (PKCß2), p22 ( phox ) and apoptosis signal-regulating kinase 1 (Ask1) expression. Diabetic dnPI3K mice displayed an exaggerated cardiomyopathy phenotype compared with diabetic Ntg mice. In contrast, diabetic caPI3K mice were protected against diastolic dysfunction, pathological cardiomyocyte hypertrophy, fibrosis and apoptosis. Protection in diabetic caPI3K mice was associated with attenuation of left ventricular superoxide generation, attenuated Anp, Bnp, PKCß2, Ask1 and p22 ( phox ) expression, and elevated AKT. Further, in cardiomyocyte-like cells, increased PI3K(p110α) activity suppressed high glucose-induced superoxide generation and enhanced mitochondrial function. CONCLUSIONS/INTERPRETATION: These results demonstrate that reduced PI3K activity accelerates the development of diabetic cardiomyopathy, and that enhanced PI3K(p110α) activity can prevent adverse cardiac remodelling and dysfunction in a setting of diabetes.

Species of Gonostomum and Paragonostomum (Ciliophora, Hypotrichida, Oxytrichidae) from the Valley of Flowers, India, with descriptions of Gonostomum singhii sp nov, Paragonostomum ghangriai sp nov and Paragonostomum minuta sp nov.

Soil samples taken from the Valley of Flowers, a component of the Nanda Devi Biosphere Reserve in the Himalayan regions of India showed the presence of twenty two free living species of ciliates. There is a preponderance of species which exhibit oral ciliature and ontogenesis in the Gonostomum pattern. Of the four species of the genus Gonostomum, three viz., G affine, G gonostomoida and G kuehnelti are similar to described species; Gonostomum singhii is new. The two species of genus Paragonostomum viz., P minuta and P ghangriai are new. The three new species are described in the present paper. All these species show prominent hypertrophied ciliary structures. Their paroral membranes reveal characteristic differences with respect to their position, number of constituent cilia and the distance between adjacent cilia. It is proposed that such species specific features of the paroral membrane have a bearing in exercising different food organism preferences as they co-exist at many sites. This single factor has possibly played an important role in species diversification of this group of hypotrichs in this isolated habitat.

Morphology and cell division of the oxytrichids Architricha indica nov. gen., nov. sp., and Histriculus histrio (Müller, 1773), Corliss, 1960 (Ciliophora, Hypotrichida).

The oxytrichid ciliate Architricha indica nov. gen., nov. sp., isolated from the river Yamuna, Delhi, shows a new combination of characters. It possesses a flexible body, 18 frontal-ventral-transverse (FVT) cirri, 3 right and 2 left marginal cirral rows, 6 dorsal bristle rows and 3 caudal cirri (CC). The FVT cirri arise from 6 primordia, which utilize 6 parental cirri in their origin as is typical of Oxytricha species. Multiple marginal rows (MMR) develop through 5 independent marginal primordia arising "within-row", 1 in each parental marginal row. All the 5 marginal rows are thus morphogenetically active. Such a mode of formation of MMR has not been recorded among oxytrichids and has necessitated separation of A. indica at the generic level. Histriculus, on the other hand, has well-known characteristics, viz. rigid body, confluent marginal rows and absence of CC. The morphogenesis of Histriculus histrio has been described by Berger and Foissner [1997. Cladistic relationships and generic characterization of oxytrichid hypotrichs (Protozoa, Ciliophora). Arch. Protistenkd. 148, 125-155]. Reinvestigation of very early stages of development revealed that (i) the FVT cirral primordia utilize kinetosomes from 5 parental FVT cirri, (ii) the primordium II of the proter is of a composite origin: kinetosomes from the oral primordium merge with the primordium II that originates from the buccal cirrus II/2 and (iii) the FVT primordia V and VI for the 2 daughter cells arise sequentially from the parental cirrus V/4. Thus, the genus Histriculus exhibits a new combination of characters with respect to the origin of FVT cirri, an additional pattern to be added to the known 6 patterns of FVT development in oxytrichids [Berger and Foissner, 1997; Berger, H., 1999. Monograph of the Oxytrichidae (Ciliophora, Hypotrichida), Kluwer Academic Publishers, Dordrecht/Boston/London].

Cisplatin induces modifications in the development of cell surface patterns of ciliates.

Cisplatin [cis-dichlorodiammineplatinum (II)] brings about significant quantitative modifications in the development of cell surface patterns in two unrelated ciliates: Stylonychia and Tetrahymena. Cells cultured in the presence of cisplatin exhibit the formation of supernumerary surface structures in the form of extra cilia/cirri (fused cilia) and other organized ciliary organelles. The metal-induced formation of extra primordia and their differentiation into supernumerary ciliary structures is governed by the normal rules of development. Additional structures are accommodated within the framework of a defined pattern, suggesting the existence of an overall global control of pattern formation. The modified pattern is rectified to its normal state during post-treatment fission cycles, suggesting the role of the cell membrane in correcting developmental errors.

Heat shock induced ultrastructural alterations in Stylonychia mytilus cells.

Fine structural observations on heat shocked cells of S. mytilus reveal that cell organelles undergo structural alterations. Mitochondria show distorted shapes with disorganized cristae and vacuolar spaces. Pulse heat shock results in dilated rough endoplasmic reticulum, abundant polysomes as well as smooth endoplasmic reticulum. Heat shocked cells show membrane bound bodies containing osmiophilic cores. In macronuclei, dense chromatin breaks up into discrete bodies accompanied by the appearance of bundles of fine filaments and clustering of nuclear pores. The most prominent changes are noticed in nucleoli. Within 15 min of heat shock, nucleoli show hypertrophy and fine fibrillar zone which gradually replaces the granular zone by 120 min giving the nucleoli ring shaped configuration. In S phase cells, macronuclei show the arrested replication band in which the diffused zone (the site of DNA replication) is absent.

Formation of a transient corticotype during excystment in Coniculostomum monilata (oxytrichidae, hypotrichida) and its modification by reorganizations.

Protargol impregnation revealed that Coniculostomum monilata, an advanced oxytrichid ciliate with the typical 18 Frontal-Ventral-Transverse (FVT) cirri but with multiple rows of right marginal cirri (RMC) and dorso-marginal cilia (DM), requires at least 3 morphogenetic cycles to acquire the normal vegetative ciliature during excystment. In the first cycle, the FVT cirral pattern is entirely different from that formed during division. The 'first ciliature' comprises 21-32 FVT cirri formed from 5 FVT primordia; these are substituted by 18 FVT cirri developed from 6 FVT primordia in the subsequent reorganization cycle. Furthermore, each successive cycle adds one RMC row and 2 DM rows while previous rows are not resorbed, unlike the FVT cirri and the left marginal cirri. Consequently, multiple RMC and DM rows develop to form a typical vegetative corticotype. Dual information for frontal ciliature appears to be a unique feature of C. monilata as a similar situation has not been observed in the other evolved oxytrichids. Presence of an alternate corticotype information (> 18 FVT cirri) possibly signifies phylogenetic relationship of C. monilata with the lower oxytrichids. Excystment morphogenesis data reaffirms the conclusion from previous investigations that C. monilata has inherited information for only 1 RMC row and 2 DM rows; multiple rows are attained through a characteristic retention of pre-existing/parental rows.

Partial retention of parental ciliature during morphogenesis of the ciliate Coniculostomum monilata (Dragesco & Njiné, 1971) Njiné, 1978 (Oxytrichidae, Hypotrichida).

Morphogenesis of Coniculostomum monilata as revealed by protargol staining shows formation of 18 Frontal-Ventral-Transverse (FVT) cirri from 6 cirral streaks. Only 3 parental FVT cirri participate in the formation of cirral streaks. While only one row of right marginal cirri (RMC) is formed during division, the non-dividing cell possesses 3-5 RMC rows. This corticotype is explained by the fact that during division parental RMC rows are retained and inwardly displaced, the innermost row being resorbed. As the cell elongates, cirri in the retained rows space out; each row is equatorially cleaved during cytokinesis. A similar retention of dorsomarginal rows occurs on the dorsal surface. Based on synapomorphies shared by C. monilata and Stylonychia mytilus, we propose that C. monilata is an evolved oxytrichid. The regular retention of ciliary structures through several generations is, however, a unique character amongst the evolved oxytrichids and may be considered as an autapomorphy to describe C. monilata. Another such feature of C. monilata is the moniliform macronucleus, unique among the advanced oxytrichids. The present study reaffirms the importance of morphogenetic criteria in ciliate systematics. The original description of C. monilata Njiné, 1978 has been rectified with respect to the nomenclature of cirri and phylogenetic status of the species.