Morphogenesis of a saline soil ciliate Urosoma salmastra (Dragesco and Dragesco-Kernéis, 1986) Berger, 1999 with notes on the phylogeny of Urosoma (Ciliophora, Hypotrichia).

Morphogenesis in a population of Urosoma salmastra collected from saline soil in Weinan, China, was investigated using protargol staining. The main morphogenetic event that characterizes U. salmastra is the inclusion of cirrus III/2 in the formation of the frontoventra-transverse cirral anlagen. We also provide small subunit ribosomal DNA gene sequences for a population of U. salmastra and for two populations of U. emarginata. The molecular phylogeny indicates that Urosoma is not monophyletic, viz, U. emarginata branches separately from its congeners and the closest relative of U. salmastra is Oxytricha granulifera.

Morphological description of three marine ciliates (Ciliophora, Scuticociliatia), with establishment of a new genus and two new species.

Three marine scuticociliates, Falcicyclidium fangi nov. gen., nov. spec., Falcicyclidium atractodes nov. spec., and Cristigera media Kahl, 1928 were investigated using live observation and silver impregnation methods. The genus Falcicyclidium is distinguished by the combination of: (i) dorsoventrally flattened body, (ii) hook-like (falciform) paroral membrane, (iii) anterior end of paroral membrane posterior to anterior end of membranelle 1, and (iv) multiple caudal cilia. Falcicyclidium fangi nov. spec., the type of the new genus, can be recognized by the combination of its large size, extremely dorsoventrally flattened (3:1) body, consistently 10 somatic kineties, and the broad, elongate buccal area occupying 60% of the body length. Falcicyclidium atractodes nov. spec. is mainly characterized by a unique spine projecting from both the anterior and posterior end. The uncommon form, Cristigera media is redescribed based on the population from Qingdao, the statistic data and additional features, especially the morphology of the living cells, are documented.

Morphological redescriptions of four marine ciliates (Ciliophora: Cyrtophorida: Dysteriidae) from Qingdao, China.

The morphology and infraciliature of four marine cyrtophorid ciliates isolated from Qingdao, China, were investigated. Based on the present work and on previous data, improved diagnoses for three rarely known species are provided: (1) Mirodysteria decora; small-sized marine Mirodysteria about 35-60 × 25-35 µm in vivo, oval in outline; body surface with two or three conspicuous dorsal spines and one caudal spine; three right kineties, the rightmost one extending dorso-apically; left frontal kineties reduced, each consisting of three basal bodies only; podite subcaudally positioned; two ventrally located contractile vacuoles. (2) Dysteria legumen; body oval with two longitudinal grooves on different plates; six right kineties, the rightmost two of which extend dorso-apically; two left frontal kineties and two ventrally located contractile vacuoles. (3) Dysteria proraefrons; body about 60×35 µm in vivo; six right kineties, the two rightmost of which extend dorso-apically and the leftmost one is considerably shortened; three left frontal kineties; two ventrally located contractile vacuoles. A population of D. derouxi with eight or nine right kineties is also briefly described. The current investigation further demonstrates high diversity and cosmopolitan distribution of this highly specialized group of benthic ciliates.

Morphological and molecular information of a new species of Geleia (Ciliophora, Karyorelictea), with redescriptions of two Kentrophoros species from China.

The morphology and infraciliature of three karyorelictean ciliates, Geleia sinica spec. nov. and two poorly known Kentrophoros species, K. flavus and K. gracilis, isolated from the intertidal zone of a beach at Qingdao, China, were investigated. Geleia sinica spec. nov. is distinguished from its congeners by the following combination of characters: body medium-sized and slender-cylindrical; with a conspicuous prebuccal fossa; 28-34 somatic kineties; about 40 short adoral polykineties; intrabuccal kinety composed of 25-34 dikinetids; paroral kineties composed of closely spaced dikinetids. The comparison with similar congeners clearly supports the validity of this new species based on morphological and small subunit (SSU) rRNA gene sequence data. In light of these new data the "well-known" morphotype, Geleia simplex (Fauré-Fremiet, 1951), is redefined. Two Kentrophoros species are redescribed and improved diagnoses are supplied. Kentrophoros flavus Raikov and Kovaleva, 1968 is mainly characterized by having about 33 macronuclei and 12 micronuclei forming a row that extends along the cell meridian, and 12-19 ciliary rows on the right side of the cell. Kentrophoros gracilis Raikov, 1963 is characterized by having about 14 macronuclei, 13 micronuclei and 10-13 kineties on the right side of the cell.

Dynamic behaviour of stationary pronuclei during their positioning in Paramecium caudatum.

During conjugation of Paramecium caudatum, there are two well-known stages when nuclear migration occurs. What happens to the nuclei is closely related to their localisations in cells. The first of these stages is the entrance of one meiotic product into the paroral region. This nucleus survives, while the remaining three outside this area degenerate. The second stage is the antero-posterior localisation of eight synkaryon division products. Four posterior nuclei are differentiated into macronuclear anlagen, whereas four anterior nuclei remain as the presumptive micronuclei. In this experiment, the process of the third prezygotic division of P. caudatum was studied with the help of protargol staining. Here, a third nuclear migration was discovered. By two spindle turnings and two spindle elongations, stationary pronuclei were positioned near migratory pronuclei. This positioning of stationary pronuclei could shorten the distance for transferred migratory pronuclei to recognise and reach the stationary pronuclei. This fosters the synkaryon formation of P. caudatum.

A practical method of Ludox density gradient centrifugation combined with protargol staining for extracting and estimating ciliates in marine sediments.

Methodological impediments have long been the main problem in estimating the ecological role of marine benthic ciliates. Percoll density centrifugation is currently the most efficient technique for extracting ciliates from fine-grained sediments, while the high cost and low density of Percoll limit its wide application. We developed a protocol of density gradient centrifugation using the cheap sol Ludox HS 40 in combination with the quantitative protargol stain (QPS) to enumerate and identify marine benthic ciliates. The combined Ludox-QPS method involves sample collection and salt reduction, extraction with Ludox centrifugation, and preparation with the QPS technique. The recovery efficiency of Ludox was first tested with azoic sandy and muddy sediments. A 94-100% recovery rate of ciliates was reached. The method was further tested with natural sandy, muddy-sand and muddy sediments. Excellent extraction efficiencies were consistently obtained: an average of 97.6% for ciliates in sand, and 96.9-97.8% for nematodes in the three types of sediments. The high efficiencies indicate that the method allows for simultaneous enumeration of micro- and meiobenthos. Advantages of the new method include: (i) reliable and cost-efficient operation; (ii) appropriate centrifugation for both micro- and meiobenthos; and (iii) applicability to large samples and routine ecological surveys.

Redescription of the tintinnid Stenosemella pacifica Kofoid and Campbell, 1929 (Ciliophora, Spirotricha) based on live observation, protargol Impregnation, and Scanning Electron Microscopy.

The tintinnid ciliate Stenosemella pacifica Kofoid and Campbell, 1929 was occasionally recorded from the pelagial of temperate, subtropical, and tropical neritic waters. Since its cytological features were unknown, the species is redescribed from material collected in the pelagial of the Irish Sea, using live observation, protargol impregnation, and scanning electron microscopy. Furthermore, the species diagnosis is improved to include new characteristics, e.g. the somatic ciliary pattern comprising a ventral, dorsal, and posterior kinety as well as a right, left, and lateral ciliary field. The stomatogenesis of S. pacifica is typical for species with such a complex somatic ciliary pattern: the oral primordium develops hypoapokinetally posterior to the lateral ciliary field. The presence of windows in the lorica collar of Stenosemella ventricosa, the type of the genus, necessitates (i) an improved genus diagnosis, (ii) a synonymization of the genus LuminellaKofoid and Campbell, 1939, and (iii) a transfer of the Luminella species to the genus Stenosemella, including Luminella neocalifornica, which becomes Stenosemella neocalifornica nov. comb. Owing to the lack of a description, Stenosemella crateri is considered a nomen nudum.

Cortical structure in non-dividing and dividing Diophrys japonica spec. nov. (Ciliophora, Euplotida) with notes on morphological variation.

The morphology and morphogenesis of Diophrys japonica spec. nov., isolated from the Mie Port, Nagasaki, Japan, were investigated from life and following impregnation with protargol. The new species is recognized by the following characters: Body elliptical in outline and slightly greyish to yellowish in color; size in vivo about 80-120 x 50-70 microm; pellicle flexible, with underlying granules densely arranged in lines; ciliature comprising about 30-46 adoral membranelles, 4-7 frontal, 1-4 ventral and 4-7 transverse cirri, always 1 left marginal and 3 caudal cirri, and 4 dorsal kineties; usually two macronuclear nodules; fragment kinety with 2-5 dikinetids; marine habitat. The main morphogenetic events are: (1) the opisthe's oral primordium develops de novo in a subsurface pouch near the left transverse cirri; (2) the proter retains the parental AZM except for reorganization of some proximal membranelles; (3) cirral anlagen for the frontal, ventral and transverse cirri in both dividers develop separately from the oral primordium or parental cirri, and are derived from the separation of primary primordia that originate de novo; (4) the anlagen for the left marginal cirrus and fragment kinety also form de novo and separately; (5) dorsal kinety anlagen occur within the parental structures at mid-body and posterior end of the cell, of which the right-most one contributes three caudal cirri from its posterior portion. Based on available ontogenetic data, the author proposes that the numbers of left marginal and caudal cirri can be regarded as reliable characters for species identification, while the numbers of frontal, ventral and transverse cirri are not consistent enough for species distinction. A key to the eleven adequately known species of Diophrys is presented.

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].

A new investigation of the marine ciliate, Trachelostyla pediculiformis (Cohn, 1866) Borror, 1972 (Ciliophora, Hypotrichida), with establishment of a new genus, Spirotrachelostyla nov. gen.

The morphology and infraciliature of the type species of the ciliate genus Trachelostyla, T. pediculiformis (Cohn, 1866) Borror, 1972, collected from the Bohai Sea near Tianjin, north China, were investigated using live observation and protargol impregnation. The Chinese population corresponds well with the original description, as follows: size about 80-150 x 20-30 microm in vivo; body dorsoventrally flattened and non-spiral; outline elongate, with anterior region conspicuously narrowed while posterior not; on average 14 macronuclear nodules, two micronuclei and 42 adoral membranelles; 16-24 cirri in left and 21-31 in right marginal row. In total 18 frontoventral-transverse cirri: constantly 11 cirri in frontal area and two ventral ones together with five enlarged transverse cirri (TC). Three inconspicuous caudal cirri (CC); six dorsal kineties with prominent cilia. The literature on T. pediculiformis is critically reviewed, showing that the descriptions so far do not agree very well. To clarify the taxonomic status of this species, the population from the Chinese coast of the Bohai Sea is designated as a neotype. The small subunit rRNA gene was sequenced for the neotype and the molecular phylogenetic tree basically supports the establishment of the family Trachelostylidae Small & Lynn, 1985. Based on the data obtained, the genus Trachelostyla Borror, 1972 is redefined thus: dorsoventrally flattened trachelostylids, body non-spirally twisted and elongate, with peristomal region conspicuously narrowed; ciliature generally in 11:2:5:3 pattern, i.e. 11 cirri in frontal region, two ventral cirri located anterior to five TC, three CC present; one left and one right row of marginal cirri not confluent posteriorly. A new genus Spirotrachelostyla nov. gen. is proposed to include the taxa which were previously assigned to Trachelostyla but characterized by spirally twisted, spindle-like body shape and a variable number of cirri in the peristomal area. Three new combinations are suggested: Spirotrachelostyla spiralis (Dragesco & Dragesco-Kernéis, 1986) nov. comb. [basionym: Trachelostyla spiralis Dragesco & Dragesco-Kernéis, 1986], S. tani (Hu & Song, 2002) nov. comb. [basionym: T. tani Hu & Song, 2002] and S. simplex (Kahl, 1932) nov. comb. [basionym: Stichotricha simplex Kahl, 1932].

Establishment of a new amphileptid genus, Apoamphileptus nov. gen. (Ciliophora, Litostomatea, Pleurostomatida), with description of a new marine species, Apoamphileptus robertsi nov. spec. from Qingdao, China.

A new pleurostomatid genus Apoamphileptus is described, which is diagnosed as: Belonging to the Amphileptidae with spica on right side; on each side of the cell, a single perioral kinety, which encircles the cytostome and does not extend to the posterior end of the cell; somatic kineties of both sides near ventral margin shortened and forming a postoral suture; two to several extra fragments with densely arranged dikinetids located in anterior portion of left side. As the type species, the morphology and infraciliature of Apoamphileptus robertsi nov. spec., isolated from a shrimp-farming pond near Qingdao (Tsingtao), China, have been investigated using living observations and the protargol silver impregnation method. The diagnosis for this new species is: Apoamphileptus 90-180 x 30-60 microm in vivo, body elongate pyriform-shaped and slightly flattened; with one cross-striated band along the cytostome; 2-6 (generally 4) large macronuclear nodules, one micronucleus; 33-43 right somatic kineties; left side 6-8 kineties; two extra anterior fragments on left side; about 13 contractile vacuoles dispersed throughout whole body; extrusomes absent or not recognizable; marine habitat. Some morphologically related morphotypes are discussed and tabulated. Regarding the pattern of infraciliature and other morphological features, the well-described fresh-water species, Amphileptus claparedii Stein, 1867 is believed to be a member of this new genus, hence a new combination is suggested: Apoamphileptus claparedii (Stein, 1867) nov. comb.

Urotricha psenneri n. sp. and Amphileptus piger (Vuxanovici, 1962) n. comb., two planktonic ciliates (Protozoa, Ciliophora) from an oligotrophic lake in Austria.

Two euplanktonic ciliates, Urotricha psenneri n. sp. (Prostomatida) and Amphileptus piger (Vuxanovici, 1962) n. comb. (Pleurostomatida), were discovered in the surface plankton of the oligotrophic Lake Traunsee in Austria. Their morphology and infraciliature were studied in live cells as well as in specimens impregnated with protargol and silver nitrate. Urotricha psenneri is a small urotrichid, less than 50 microm length and with a single caudal cilium. It is unique in having (i) a massive oral basket projecting as a conspicuous bulge with cylindrical microfibrillar annulus and (ii) a curved brosse row 1 in the broad, barren circumoral area. Amphileptus piger (Vuxanovici, 1962) is about 55 x 13 microm in vivo, has two macronuclear nodules with a single micronucleus in between in the posterior body half, has a single contractile vacuole with a terminal excretory pore, and few, but thick and thus highly conspicuous extrusomes. The amphileptid ciliary pattern (spica) is difficult to recognise due to the widely spaced basal bodies.

[Study on the activity of fibroblast in hypertrophic scar].

To determine the state of fibroblast during the process of development of hypertrophic scar (HS), 40 specimens of HS in different periods were collected. The expressions of prolifrating cell nuclear antigen (PCNA) and Ag-protein in nucleolar organizer regions (Ag NORs) as well as the content of total amino acids in the tissues were examined. The hypertrophic scar of 1st and 3rd month old, the expression of PCND and Ag NORs were the highest. In the 9th and 12th month old, althrough PCNA was nearly negative, but the expression of Ag NORs was low. The content of total amino acid was increased gradually as HS developed but the increase of amount of hydroxyproline was markedly slowed down in 9 month old HS. It was suggested that: (1) in the developing process of HS the proecess of overproliferation of fibroblasts was short and limitted in 1-3 months period in the process of wound lealing; (2) the synthesis of collagen was nearly stopped at 6 months, but that of other extracellular matrix such as fibronectin and proteoglycan might be continued to aggregate after 12 months.

The expression of tyrosinase, tyrosinase-related proteins 1 and 2 (TRP1 and TRP2), the silver protein, and a melanogenic inhibitor in human melanoma cells of differing melanogenic activities.

The expression of various melanogenic proteins, including tyrosinase, the tyrosinase-related proteins 1 (TRP1) and 2 (TRP2/DOPAchrome tautomerase), and the silver protein in human melanocytes was studied in six different human melanoma cell lines and compared to a mouse derived melanoma cell line. Analysis of the expression of tyrosinase, TRP1, TRP2, and the silver protein using flow cytometry revealed that in general there was a positive correlation between melanin formation and the expression of those melanogenic enzymes. Although several of the melanoma cell lines possessed significant activities of TRP2, the levels of DOPAchrome tautomerase in extracts of human cells were relatively low compared to those in murine melanocytes. Melanins derived from melanotic murine JB/MS cells, from melanotic human Ihara cells and HM-IY cells, from sepia melanin, and from C57BL/6 mouse hair were chemically analyzed. JB/MS cells, as well as Ihara cells and HM-TY cells, possessed significant amounts of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) derived melanins, this being dependent on the activity of TRP2. Kinetic HPLC assays showed that 5,6-dihydroxyindole (DHI) produced during melanogenesis was metabolized quickly to melanin in pigmented KHm-1/4 cells, whereas DHI was stable in amelanotic human SK-MEL-24 cells. A melanogenic inhibitor that has been purified from SK-MEL-24 cells that suppressed oxidation of DHI in the presence or absence of tyrosinase, but had no effect on DHICA oxidation. The sum of these results suggests that the expression of melanogenic enzymes as well as the activity of a melanogenic inhibitor are critical to the production of melanin synthesis in humans.

Incorporation of argyrophilic proteins into the nucleolus-associated chromatin caused by exposure to actinomycin D.

The nucleolus-associated chromatin (NAC) became argyrophilic when the root tips of Vicia faba were treated with 0.2 microgram/ml actinomycin D (AMD) for 15 hr at 22 degrees C. To determine how the argyrophilic NAC is formed, detailed observations were carried out on thin sections following silver staining and ribonucleoprotein (RNP) preferential staining. In a few instances, thick channels with loosely packed fibrillar material (light fibrillar area) were seen meandering throughout the nucleoli. Large granules 280-330 A in diameter (300 A granules), which responded positively to both RNP preferential staining and silver staining, were sometimes present within the light fibrillar area. Some fragments of NAC were also seen in the light fibrillar area and, interestingly, they responded positively to silver even though they were bleached by the RNP preferential staining. Most nucleoli showed a segregation of two major components, the fibrillar and granular components. The nucleolar vacuoles were localized strictly within the fibrillar component and usually accompanied the light fibrillar area or, alternatively, contained a number of 300 A granules. Another category was characterized by the presence of poorly developed vacuoles in which the 300 A granules were still found, and the spherical argyrophilic NAC associated with the surface of the nucleolus. The present observations suggest that AMD induces the DNA-containing structure engaged in ribosomal RNA transcription to condense, and during this process some argyrophilic proteins are incorporated into the chromosomal structure thereby causing the NAC to become argyrophilic. In this context, the NAC is assumed to contain ribosomal RNA genes.