Peripheral Nucleus Research Articles

T세포와 양방향 작용을 통한 마우스 림프절로부터 분리된 fibroblastic reticular cell의 기능적 분석

Fibroblastic reticular cells (FRC)는 림프절 T세포 지역에 구조적 골격 형성을 하며 유입 T 세포의 안내길을 제공한다. FRC는 림프절에서 T세포 생물학 발달에 기여한다. 따라서, 이것이 FRC와 T세포 사이에서 FRC의 세포생물학적 근본 기능을 알아보게 하였다. FRC 배양 상등액은 T세포 사멸을 저해하였다. FRC 상등액은 doxorubicin에 대하여 T세포에 Bcl-xL의 발현을 증가시켰다. FRC와 T세포의 공배양은 FRC의 액틴 골격의 변화와 형태적 변화를 유도하였다. 또한, FRC와 T세포의 공배양은 T 세포가 FRC 단일층에 부착하는 결과를 유도하였고 막결합형 intercellular adhesion molecule (ICAM)-1 단백질의 발현은 약간 증가한 반면 용해성 ICAM-1 (sICAM-1) 발현은 시간 의존적으로 드라마틱하게 증가하였다. FRC는 T세포에 의해 분비되는 tumor necrosis factor (TNF) 패밀리들에 의해 CCL5, CXCL1, CXCL5, CXCL16, CCL8, CXCL13와 같은 케모카인들과 ICAM-1 그리고 MMPs의 발현량을 증가시켰다. <TEX>$TNF{\alpha}$</TEX>가 FRC에 처리 되었을때 <TEX>$NF{\kappa}B$</TEX> canonical pathway의 RelA는 핵으로 전좌 되었지만, agonistic anti-<TEX>$LT{\beta}R$</TEX> antibody로 처리된 FRC에서 non-canonical <TEX>$NF{\kappa}B$</TEX> pathway의 RelB의 카운터 파트너인 p100의 분해산물 p52는 핵주변부로 축적되었다. 결론적으로 FRC는 FRC와 T세포 양방향 협력을 통해 T세포 생물학적 기능을 증진한다. 이러한 상호협력 관계는 면역반응 동안 조직의 통합성과 기능을 유지하는데 도움을 줄 것으로 사료된다. Fibroblastic reticular cells (FRCs) form the structural backbone of the T zone provide a guidance path for immigrating T cells in the lymph node (LN). FRCs may contribute directly to developing T-cell biology in the LN and allow analyses of fundamental aspects of FRC biology related to T cells. FRCs inhibited T-cell apoptosis, and FRC culture supernatants strongly induced the expression of Bcl-xL in T cells against doxorubicin. Coculture of FRC and T cells resulted in rearrangements of the actin cytoskeleton, as well as global changes in the morphology of the FRCs. In addition, when cocultured, the T cells adhered to the FRC monolayer, and the membrane intercellular adhesion molecule (ICAM)-1 was slightly increased by day-dependent manner. In contrast, the expression of soluble ICAM-1 was dramatically increased in a day-dependent manner. Several chemokines, such as CCL5, CXCL1, CXCL5, CXCL16, CCL8, CXCL13, and ICAM-1, and MMPs were expressed in FRCs sensed by tumor necrosis factor (TNF) families. Nuclear factor kappa B (<TEX>$NF{\kappa}B$</TEX>)-RelA of the <TEX>$NF{\kappa}B$</TEX> canonical pathway was translocated into FRC nuclear by <TEX>$TNF{\alpha}$</TEX>. In contrast, p52 proteolyzed from p100, a counterpart of RelB of the noncanonical <TEX>$NF{\kappa}B$</TEX> pathway, accumulated in the peripheral FRC nucleus by agonistic anti-<TEX>$LT{\beta}R$</TEX> antibody. In summary, we propose a model in which FRCs engage in bidirectional crosstalk to increase the efficiency of T-cell biology. This cooperative feedback loop may help to maintain tissue integrity and function during immune responses.

Genetic diversity, structure and conservation of the endangered Cantabrian Capercaillie in a unique peripheral habitat

Populations at the rear edge of the species’ range are often at a high risk of extinction due to their isolation, fragmentation and small population sizes. However, these populations also play a relevant role in the conservation of biodiversity since they may represent a valuable genetic resource. The endangered Cantabrian Capercaillie (Tetrao urogallus cantabricus) inhabits deciduous forests of the Cantabrian Mountains of Spain, at the southwestern limit of the species’ range. Recently, nine Cantabrian Capercaillie leks were discovered in Pyrenean oak forests of the southern slope of the Cantabrian range, where the subspecies historically occurred. To elucidate if the origin of this peripheral population nucleus is the result of a historical range contraction or a recent re-colonization from the core population, we sampled moulted feathers from all the known leks in the southern peripheral forests and from the adjacent main core population, based on nine microsatellite loci genotypes. No significant genetic differentiation was detected between main core and peripheral forests suggesting that gene flow is not interrupted between these nuclei. Contrary to expected, peripheral forests did not represent sink populations, since gene flow mainly occurred from southern peripheral to northern main core forests. Therefore, the origin of these birds inhabiting the peripheral nucleus seems not to be a recent colonization but relicts from the former distribution range that have remained unnoticed in a drier and warmer environment than described so far for the species. Cantabrian Capercaillie faces a high risk of extinction in the southernmost forests of its distribution, not only because of its peripheral location but also due to its small population size, low genetic diversity and low incoming gene flow. According to our results, this peripheral nucleus could represent an expanding edge for the population if Pyrenean oak forests continue to spread out southwards and consequently stress the need for conservation programs to preserve habitat availability and forest connectivity.

Size of the emission source and collectivity in ultra-relativistic p–Pb collisions

The interferometric radii in the system formed in ultra-relativistic proton–lead collisions are investigated in a framework based on event-by-event (3+1)-dimensional viscous hydrodynamics. We argue that the most central p–Pb collisions undergoing collective expansion behave similarly to the peripheral nucleus–nucleus collisions. The interferometric observables can serve as signatures of the formation of an extended fireball.

Exclusive ${\rm{D\bar D}}$ meson pair production in peripheral ultrarelativistic heavy ion collisions

The cross sections for exclusive D + D − and D 0 ¯ D0 meson pair production in peripheral nucleus - nucleus collisions are calculated and several differential distributions are presented. The calculation of the elementary ! D ¯ D cross section is done within the heavy-quark approximation and in the Brodsky- Lapage formalism with distribution amplitudes describing recent CLEO data on leptonic D + decay. Absorption effects are discussed and quantified. The cross sections of a few nb are predicted for RHIC and of a few hundreds of nb for LHC with details depending on the approximation made in calculating elementary ! D ¯ D cross sections.

Production of exotic nuclei in peripheral nucleus–nucleus collisions below 10 A MeV

Abstract Experimental data from the literature concerning nucleus–nucleus collisions at beam energies of few A MeV above the Coulomb barrier are investigated in this work with emphasis on projectile-like fragment distributions. In peripheral collisions at beam energies below 10 A MeV, deep-inelastic transfer is shown to be the dominant reaction mechanism. In addition, the mechanism may involve an extension of the nuclear profile in the window (neck) region, observed primarily in reactions with very heavy target nuclei. Isoscaling observed at these energies can be used as a tool to predict the production rates of exotic nuclei in reactions induced by exotic secondary beams.

Exclusive [formula omitted] meson pair production in peripheral ultrarelativistic heavy-ion collisions

The cross sections for exclusive D+D− and D0D¯0 meson pair production in peripheral nucleus–nucleus collisions are calculated and several differential distributions are presented. The calculation of the elementary γγ→DD¯ cross section is done within the heavy-quark approximation and in the Brodsky–Lapage formalism with distribution amplitudes describing recent CLEO data on leptonic D+ decay. Realistic (Fourier transform of charge density) charge form factors of nuclei are used to generate photon flux factors. Absorption effects are discussed and quantified. The cross sections of a few nb are predicted for RHIC and of a few hundreds of nb for LHC with details depending on the approximation made in calculating elementary γγ→DD¯ cross sections.

Protective effects of puerarin on lens epithelial cells in rat diabetic cataract

The therapeutic effects of general and local applications of puerarin in the treatment of streptozotocin (STZ)-induced rat diabetic model were compared. Experimental research. We equally divided normal Sprague-Dawley (SD) rats into a STZ group, a peritoneal injection group, a peribulbar injection group and a control group. STZ, peritoneal injection and peribulbar injection groups were first treated with STZ. Subsequently, the STZ group was injected with normal saline intraperitoneally, while in the later two groups puerarin was injected through peritoneal and peribular routes, respectively. Control group only received peritoneal injection of saline. The morphology of lens epithelial cells (LEC) and their subcellular structure were examined by bright-field microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) 20, 40 and 60 days after the injection. Nitrogen oxide (NO) and nitric oxide synthase (NOS) were measured by biochemistry methods. Finally, inducible nitric oxide synthase (iNOS) protein and mRNA levels were monitored by Western blot and RT-PCR, respectively. Data was processed with two factorial experiment analysis of variance. Twenty to sixty days after the injection, marked or complete lens opacities appeared in the STZ group, whereas only slight opacities appeared in the lens in peritoneal and peribulbar puerarin groups and the lens in the control group remained clear. At the 20th, 40th and 60th day after the injection, optical microscope detected pathological changes of LEC in the STZ group. The cell volume was decreased with a dense nucleus and many bubbles appeared around the equator area. Under TEM, enlargement of cell gap, vacuoles in the cytoplasm, swelling of mitochondria and unclear structure of rough endoplasmic reticulum appeared in the LEC of the STZ group. Part of the nucleus was in karyopyknosis and peripheral nucleus gap was enlargement. Under SEM, normal fiber conjunction structure of the lens disappeared, fibers were swelling, part of fiber membranes were discontinuous, detached, and accumulated in certain areas. Mild lens opacities detected by bright-field microscope were developed in peritoneal and peribulbar puerarin injection groups. Nucleus and fibers in the lens cells of both groups appeared to be normal, with minor swelling of mitochondria, minor enlargement of endoplasmic reticulum and slight increase of intracellular space. NO, NOS and iNOS protein and mRNA of the lens were increased and up-regulated in STZ group. In the other two groups only minor changes were present and the changes were significantly less than that of the STZ group but greater than that in the control group. Peritoneal and peribulbar injection of puerarin have similar therapeutic effects in the treatment of rat diabetic cataract.

Comparative ultrastructural analysis of diestrous and anestrous canine Grade 1 cumulus-oocyte complexes

The objective of the present study was to characterize, by means of transmission electron microscopy, immature Grade 1 cumulus-oocyte complexes (COCs) obtained from ovaries collected from bitches at diestrus and anestrus, after routine ovariohysterectomy. Cumulus-oocyte complexes were recovered after slicing the ovarian cortex and Grade 1 COCs were selected and prepared for transmission electron microscopy. All oocytes were at germinal vesicle stage in two different configurations. In 29 out of 37 COCs, oocytes presented a central or eccentric located nucleus (GVc), frequently containing a reticulated nucleolus, with a predominance of profound follicular cell processes (FCP), abundant endoplasmic reticulum (ER) and a moderated number of lipid droplets. In eight out of 37 COCs, oocytes had a peripheral located nucleus (GVp) containing small compact nucleolus, a thin perivitelline space, both superficial and profound FCP, moderate ER content and abundant lipid droplets. A higher ( p < 0.05) proportion of GVp was found in oocytes collected in diestrus (36.8% – 7 out of 19) than in anestrus (5.6% – 1 out of 18). In addition, distinct ultrastructure characteristics among GVc associated with estrous phase were noted. Furthermore, a number of structures, some of which had not been reported before, were present in canine prophase I oocytes at variable frequencies. The evident variation in the presence, quantity and distribution of cell organelles in canine immature Grade 1 oocytes is discussed in relation to the biological phase of the oocyte and the reproductive stage of the donor bitch.

Vitellogenesis of basal trematode Aspidogaster limacoides(Aspidogastrea: Aspidogastridae)

The vitellogenesis of the trematode Aspidogaster limacoides (Aspidogastrea: Aspidogastridae), a parasite of cyprinid fishes, is described here using transmission electron microscopy. Four different stages of vitellocytes are differentiated: immature vitellocytes, early maturing vitellocytes, advanced maturing vitellocytes and mature vitellocytes. The process follows the same general pattern already described in other free-living neoophorans and parasitic flatworms (i.e. Trematoda, Monogenea and Cestoda): differentiation into mature vitelline cells involves the development of mitochondria, granular endoplasmic reticulum, Golgi complexes, lipid droplets and shell-globules. Mature vitellocytes of A. limacoides are composed of numerous shell-globule clusters, few lipid droplets and glycogen granules. They differ from those of another aspidogastrean Rugogaster hydrolagi in that they possess numerous globules tightly packed and by the presence of only one type of vitelline material. The interstitial tissue of vitelline follicles of A. limacoides contains a peripheral nucleus and long cytoplasmic projections extending between vitelline cells. Since aspidogastreans are considered as an archaic group of parasitic flatworms and thus have a strategic phylogenetic position, future works needs to pay special attention to the ultrastructural and chemical composition of mature vitellocytes within this basal group of trematodes.

51 DELAYED NUCLEOLOGENESIS IN PORCINE PREIMPLANTATION EMBRYOS DEVELOPED IN VIVO AND PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

Nucleolus is known to be a well-suited morphological marker for embryo technologies. Failure in de novo nucleolar formation during embryonic genome activation (EGA) has been observed in many species. The aim of the present study was to investigate nuclear changes and nucleolar formation during EGA in the porcine preimplantation embryos developed in vivo and produced by somatic cell nuclear transfer (SCNT). Embryos were collected at early and late 1-cell stage, 2-, 4-, and 8-cell stage, early and late blastocyst stage, fixed in 3% glutaraldehyde for 1 h, and processed for transmission electron microscopy. In vivo embryos from 1- and 2-cell stages showed electron dense, spherical nucleolar precursor bodies (NPB) in centrally located nuclei with well-developed nuclear envelope and condensed chromatin. Two 1-cell-stage embryos, however, had represented metaphase chromosomes in the periphery. At the 4-cell stage, in vivo embryos displayed fibrillo-granular nucleoli containing all 3 functional nucleolar compartments: fibrillar centers (FC), dense fibrillar component (DFC), and granular component (GC). The nuclei were centrally located, round, and had complete nuclear envelopes. The same types of nuclei and nucleoli were observed for all following stages. On the other hand, embryos produced by SCNT at early 1-cell stage showed centrally located, irregular-shaped nuclei with incomplete nuclear envelopes and condensed chromatin with large intact NPB. Exceptionally, 1 out of the 5 embryos presented a peripheral nucleus with partially condensed chromatin lacking nuclear envelope and fibrillo-granular nucleolus probably persisting from donor fibroblast. Only 2 out of 5 late-1-cell SCNT embryos showed nuclear structures. The nuclei had irregular shapes, complete nuclear membranes, and contained large NPB. At the 2- and 4-cell stages, the embryos presented central nuclei with complete nuclear envelopes. Some of the embryos showed more than one nucleus of varying shapes and sizes. The fibrillo-granular nucleoli were first observed toward the 8-cell stage. The embryos from this stage contained irregularly shaped nuclei with well-developed nuclear envelopes. The nucleoli displayed fibrillar and granular compartments in SCNT 8-cell stage embryos, but lacked the typically structured functional nucleoli observed in in vivo embryos. The absence of formation of functional nucleoli at the 4-cell stage and altered nuclear ultrastructure during the EGA in SCNT embryos, thus, may be one of the main reasons for decreased developmental competence of SCNT embryos.

Influence of the nucleon–nucleon collision geometry on the determination of the nuclear modification factor for nucleon–nucleus and nucleus–nucleus collisions

The influence of the underlying nucleon–nucleon collision geometry on evaluations of the nuclear overlap function (TAB) and number of binary collisions (Ncoll) is studied. A narrowing of the spatial distribution of the hard-partons with large light-cone fraction x in nucleons leads to a downward correction for Ncoll and TAB, which in turn, results in an upward correction for the nuclear modification factor RAB. The size of this correction is estimated for several experimentally motivated nucleon–nucleon overlap functions for hard-partons. It is found to be significant in peripheral nucleus–nucleus and nucleon–nucleus collisions, and is much larger at the LHC energy of s=5.5 TeV than for the RHIC energy of s=200 GeV. The implications for experimental measurements are also discussed.

Cellular Epigenetic Modifications of Neural Stem Cell Differentiation

Emerging information indicates that epigenetic modification (i.e., histone code and DNA methylation) may be integral to the maintenance and differentiation of neural stem cells (NSCs), but their actual involvement has not yet been illustrated. In this study, we demonstrated the dynamic nature of epigenetic marks during the differentiation of quiescent adult rat NSCs in neurospheres. A subpopulation of OCT4(+) NSCs in the neurosphere contained histone marks, trimethylated histone 3 on lysine 27 (3me-H3K27), 2me-H3K4, and acetylated H4 (Ac-H4). A major decrease of these marks was found prior to or during differentiation, and was further diminished or reprogrammed in diverse subpopulations of migrated NSCs expressing nestin or beta-III-tubulin. The DNA methylation mark 5-methyl-cytosine (5-MeC), and DNA methyltransferase (DNMT) 1 and 3a expression also correlated to the state of differentiation; they were highly present in undifferentiated NSCs but downregulated in migrated populations. In contrast, DNA methyl-CpG-binding protein (MBD1) was low in undifferentiated NSCs in neurospheres, but highly appeared in differentiating NSCs. Furthermore, we found an outward translocation of DNA methylation marker 5-MeC, DNMT1, DNMT3a, and MBD1 in NSCs as differentiation began and proceeded; 5-MeC from homogeneous nucleus to peripheral nucleus, and DMNT1a and 3a from nuclear to cytoplasm, indicating chromatin remodeling. Treatment with DNA methylation inhibitor, 5-aza-cytidine, altered DNA methylation and disrupted migration as indicated by a reduction of migrated neurons and differentiation. These results indicate that chromatin is dynamically remodeled when NSCs transform from the quiescent state to active growth, and that DNA methylation modification is essential for neural stem cell differentiation.

Pharmacological enhancement of disc diffusion and differentiation of healthy, ageing and degenerated discs

Pharmacological enhancement of disc diffusion and differentiation of healthy, ageing and degenerated discs

Rhodaphanes brevistipitata gen. et sp. nov., a new member of the Stylonematophyceae (Rhodophyta)

J.A. West, G.C. Zuccarello, J.L. Scott, K.A. West and U. Karsten. 2007. Rhodaphanes brevistipitata gen. et sp. nov., a new member of the Stylonematophyceae (Rhodophyta). Phycologia 46: 440–449. DOI: 10.2216/07-03.1Rhodaphanes brevistipitata gen. and sp. nov. was isolated into culture as an epiphyte on the red alga Spermothamnion cymosum from a collection made at Point Lonsdale, Victoria, Australia. It is morphologically and cytologically very similar to Stylonema species. It is short (< 1.2 mm), clavate, uniseriate to multiseriate, usually unbranched and with a short stipe. Each cell has one purple-grey multilobed plastid with a central pyrenoid and peripheral nucleus. Reproduction is by release of vegetative cells (archeospores) individually or when the whole thallus gelatinises. Free spores observed in time-lapse videomicroscopy are round (7–12 μm) and glide without substratum contact. Molecular analysis of a partial sequence of the nuclear-encoded small subunit ribosomal RNA gene and plastid encoded photosystem II reaction centre protein D1 (psbA) placed Rhodaphanes in the Stylonematales, but more closely related to the genera Bangiopsis and Purpureofilum than to Stylonema. Ultrastructural examination of archeospores revealed a rough endoplasmic reticulum (RER) – Golgi association typical of the Stylonematophyceae and plastids lacking a peripheral limiting thylakoid, a feature not seen in any other genus of the class. The low–molecular weight carbohydrates digeneaside, sorbitol and trehalose are present.

Morfología y citoquímica de cultivos celulares de Aedes aegypti (Diptera: Culicidae) y susceptibilidad a Leishmania panamensis (Kinetoplastida: Trypanosomatidae)

Morphology and cytochemistry of Aedes aegypti's cell cultures (Diptera: Culicidae) and susceptibility to Leishmania panamensis (Kinetoplastida: Trypanosomatidae). The first cellular line of Aedes aegypti was developed by Grace in 1966; afterwards, other cellular lines of this species have been generated. These have been used for the study of pathogenic organisms like viruses, bacteria and parasites, which demonstrates their importance in biomedical applications. This research describes, for the first time, some cytochemical characteristics of A. aegypti cell cultures, that were infected with (MHOM/CO/87CL412) strain of Leishmania panamensis. A morphological study of the cell culture was also carried out. Maintenance of the cell culture, parasites and infection in vitro were carried out in the Laboratory of Entomology, Cell Biology and Genetics of the Universidad de La Salle. The cell cultures infected with the parasite were maintained in a mixture of mediums Grace/L15, supplemented with 10 % fetal bovine serum (FBS) at pH 6.8 and a temperature of 26 degrees C, during 3, 6 and 9 post-infection days. After this, these cell cultures were processed through High Resolution Light Microscopy (HRLM) and Transmission Electron Microscopy (TEM) based on standard protocols defined by the Group of Microscopy and Image Analyses of the Instituto Nacional de Salud. Semi-fine slices of 1 microm colored with toluidine blue were used for the morphological analysis of the culture, and ultra fine cuts of 60 to 90 nm stained with uranyl acetate and lead citrate where used for the ultrastructural study. In addition, PAS and peroxidase staining was carried out in cells fixed with methanol. The morphometric study was analyzed with software ImageJ (NIH). In the semi-fine slices, small cells were observed showing fibroblastic appearance 10.84 +/- 2.54 microm in length and 5.31 +/- 1.26 microm wide; other cells had epithelial appearance with a great peripheral nucleus, voluminous and vacuolated cytoplasm, 23.04 +/- 4.00 microm in length and 13.96 3.70 microm wide. These last ones predominated over the ones with fibroblastic appearance. Regarding the PAS coloration, 7.08% of the cells presented abundant PAS positive cytoplasmatic granules which indicated polysaccharides presence. The peroxidase test gave a negative result. The greatest percentage of infection (18.90%) of one total of 101 cells, turned up by day 6. Some cells analyzed by TEM presented a vacuolated aspect cytoplasm; some contained parasites, other fibrillar material and others were empty. The results indicate that A. aegypti cell culture can support the internalization and transformation of the parasite, which demonstrates the capacity that these cell cultures have to be infected with L. panamensis and to maintain the infection for approximately one week.

Peculiar behavior of distinct chromosomal DNA elements during and after development in the dicyemid mesozoan Dicyema japonicum

The dicyemid mesozoans are obligate parasites that inhabit the cephalopod renal appendage. Dicyemids have a simple body, consisting of approximately 30 cells: one long cylindrical axial cell contains intracellular stem cells (called axoblast), from which embryos are derived, and is surrounded by some 30 peripheral somatic cells. Somatic cells divide at most eight times in their life span, and never divide after differentiation. During early somatic cell development, numerous unique DNA sequences are first amplified and then eliminated, in the form of extrachromosomal circular DNA, leading to genome reduction. In this study we demonstrate that the remaining sequences, single-copy genes and repetitive sequences, have very different fates. Single-copy genes, such as beta-tubulin, are initially amplified, presumably via endoreduplication, but subsequently decrease in copy number through development, suggesting that the whole genome is initially amplified and then the amplified DNAs are simply diluted in successive cell divisions, with little DNA replication. In contrast, repetitive sequences are maintained even in terminally differentiated somatic cell nuclei. Considering the increasing intensity of in-situ hybridization, incorporation of BrdU, and a general correlation between nuclear content and cell size, those repetitive sequences must be selectively endoreplicated in the peripheral cell nucleus, concomitant with the increase of cell size. The biological significance of this mechanism is discussed as a unique dicyemid adaptation to parasitism.

Regulation of dynamic events by microfilaments during oocyte maturation and fertilization

Actin filaments (microfilaments) regulate various dynamic events during oocyte meiotic maturation and fertilization. In most species, microfilaments are not required for germinal vesicle breakdown and meiotic spindle formation, but they mediate peripheral nucleus (chromosome) migration, cortical spindle anchorage, homologous chromosome separation, cortex development/maintenance, polarity establishment, and first polar body emission during oocyte maturation. Peripheral cortical granule migration is controlled by microfilaments, while mitochondria movement is mediated by microtubules. During fertilization, microfilaments are involved in sperm incorporation, spindle rotation (mouse), cortical granule exocytosis, second polar body emission and cleavage ring formation, but are not required for pronuclear apposition (except for the mouse). Many of the events are driven by the dynamic interactions between myosin and actin filaments whose polymerization is regulated by RhoA, Cdc42, Arp2/3 and other signaling molecules. Studies have also shown that oocyte cortex organization and polarity formation mediated by actin filaments are regulated by mitogen-activated protein kinase, myosin light-chain kinase, protein kinase C and its substrate p-MARKS as well as PAR proteins. The completion of several dynamic events, including homologous chromosome separation, spindle anchorage, spindle rotation, vesicle organelle transport and pronuclear apposition (mouse), requires interactions between microfilaments and microtubules, but determination of how the two systems of the cytoskeleton precisely cross-link, and which proteins link microfilaments to microtubules to perform functions in eggs, requires further studies. Finally, the meaning of microfilament-mediated oocyte polarity versus embryo polarity and embryo development in different species (Drosophila, Xenopus and mouse) is discussed.

Dermocystidium sp. infection in cultured juvenile southern catfish Silurus meridionalis in China

A species of Dermocystidium was discovered on the skin and fins of reared southern catfish Silurus meridionalis Chen. The parasite only appeared and caused disease in juvenile catfish at a water temperature of 18 to 23 degrees C. Marked external macroscopical clinical signs of the disease were sluggish movement of the fish, and the appearance of white filiform dermal cysts varying in size (3-20 mm in length and 0.15-0.35 mm in width). Examination of both fixed and fresh mounts for light microscopy and of samples for transmission electron microscopy (TEM) showed spherical spores (3.2-15 microm in diameter) with a peripheral nucleus (1.1-1.8 x 0.5-1.6 microm in diameter) and a prominent refractile body (2.08-10.83 microm in diameter) which occupied most of the volume of a mature spore. Three types of spore were identified, and are presumed to represent various developmental stages. Meanwhile, TEM showed the remnant nuclei in the residual plasmodium of a cyst, revealing its degenerative process. This paper represents the first observation and description of Dermocystidium sp. parasitizing catfishes.

Complete development of Cryptosporidium parvum in host cell-free culture

The present study describes the complete in vitro development of Cryptosporidium parvum (cattle genotype) in RPMI-1640 maintenance medium devoid of host cells. This represents the first report in which Cryptosporidium is shown to multiply, develop and complete its life cycle without the need for host cells. Furthermore, cultivation of Cryptosporidium in diphasic medium consisting of a coagulated new born calf serum base overlaid with maintenance medium greatly increased the total number of Cryptosporidium stages. Type I and II meronts were detected giving rise to two morphologically different merozoites. Type I meronts, which appear as grape-like clusters as early as 48 h post culture inoculation, release merozoites, which are actively motile, and circular to oval in shape. Type II meronts group in a rosette-like pattern and could not be detected until day 3 of culturing. Most of the merozoites released from type II meronts are generally spindle-shaped with pointed ends, while others are rounded or pleomorphic. In contrast to type I, merozoites from type II meronts are less active and larger in size. Sexual stages (micro and macrogamonts) were observed within 6–7 days of culturing. Microgamonts were darker than macrogamonts, with developing microgametes, which could be seen accumulating at the periphery. Macrogamonts have a characteristic peripheral nucleus and smooth outer surface. Oocysts at different levels of sporulation were seen 8 days post culture inoculation. Cultures were terminated after 4 months when the C. parvum life cycle was still being perpetuated with the presence of large numbers of excysting and intact oocysts. Culture-derived oocysts obtained after 46 days p.i. were infective to 7- to 8-day-old ARC/Swiss mice. The impact of C. parvum developing in cell-free culture is very significant and will facilitate many aspects of Cryptosporidium research.

Complete development of Cryptosporidium parvum in host cell-free culture

The present study describes the complete in vitro development of Cryptosporidium parvum (cattle genotype) in RPMI-1640 maintenance medium devoid of host cells. This represents the first report in which Cryptosporidium is shown to multiply, develop and complete its life cycle without the need for host cells. Furthermore, cultivation of Cryptosporidium in diphasic medium consisting of a coagulated new born calf serum base overlaid with maintenance medium greatly increased the total number of Cryptosporidium stages. Type I and II meronts were detected giving rise to two morphologically different merozoites. Type I meronts, which appear as grape-like clusters as early as 48 h post culture inoculation, release merozoites, which are actively motile, and circular to oval in shape. Type II meronts group in a rosette-like pattern and could not be detected until day 3 of culturing. Most of the merozoites released from type II meronts are generally spindle-shaped with pointed ends, while others are rounded or pleomorphic. In contrast to type I, merozoites from type II meronts are less active and larger in size. Sexual stages (micro and macrogamonts) were observed within 6–7 days of culturing. Microgamonts were darker than macrogamonts, with developing microgametes, which could be seen accumulating at the periphery. Macrogamonts have a characteristic peripheral nucleus and smooth outer surface. Oocysts at different levels of sporulation were seen 8 days post culture inoculation. Cultures were terminated after 4 months when the C. parvum life cycle was still being perpetuated with the presence of large numbers of excysting and intact oocysts. Culture-derived oocysts obtained after 46 days p.i. were infective to 7- to 8-day-old ARC/Swiss mice. The impact of C. parvum developing in cell-free culture is very significant and will facilitate many aspects of Cryptosporidium research.