Differential in vitro effects of chemotherapeutic agents on primary cultures of human ovarian carcinoma.

The treatment of ovarian cancer principally relies on the use of platinum and taxane chemotherapeutic agents. Short-term clinical results have been encouraging, but long-term responses remain limited. In this report, an in vitro assay system that utilizes cells grown from human tumor explants has been used to quantitatively evaluate responses to relevant concentrations of alternative chemotherapeutic agents. The results suggest that there are significant differences in the responses of explant-derived cultured cells to the different agents tested. In an evaluation of 276 primary ovarian cancer specimens, five nonstandard drugs were tested in 51 cases. Of these 51 cases, cyclophosphamide had the highest rate of response at 67%, followed by doxorubicin at 61%, gemcitabine at 49%, etoposide at 48%, and topotecan at 14%. Venn diagrams, representing the in vitro responses to the platins and taxanes, as well as the responses to the nonstandard drugs, illustrate that there clearly are distinct differences among patients in a given population. These data underscore the potential importance of evaluating each patient's response to a number of different drugs to optimize the therapeutic decision-making process.

Nucleolar ultrastructure and protein allocation in in vitro produced porcine embryos.

The nucleolus formation was studied as an indirect marker of the ribosomal RNA (rRNA) genes activation in porcine embryos following oocyte maturation, fertilization, and culture in vitro. Nucleologenesis was assessed by transmission electron microscopy (TEM), light microscopical autoradiography following 20 min of 3H-uridine incubation, and immunocytochemical localization of key nucleolar proteins involved in rRNA transcription (upstream binding factor (UBF), topoisomerase I, and RNA polymerase I) and processing (fibrillarin, nucleophosmin, nucleolin) by confocal laser scanning microscopy. During the first four post-fertilization cell cycles, TEM revealed spherical nucleolus precursor bodies (NPBs), consisting of densely packed fibrils, as the most prominent intra-nuclear entities of the blastomeres. Fibrillo-granular nucleoli were observed in some blastomeres in a single embryo during the 5th cell cycle, i.e., the tentative 16-cell stage, where formation of fibrillar centres (FC), a dense fibrillar component, and a granular component on the surface of the NPBs was seen. In this embryo, autoradiographic labeling was detected over the nucleoplasm and in particular over the nucleoli. Fibrillarin was immunocytochemically localized in the presumptive NPBs of the pronuclei. This protein was again localized to the presumptive NPBs together with nucleolin from late during the 3rd cell cycle, i.e., the four-cell stage in some embryos. UBF, RNA polymerase I, and nucleophosmin were localized to the presumptive NPBs in a proportion of the embryos at the 4th cell cycle, i.e., the tentative eight-cell stage and onwards. Toposiomerase I was not localized to intra-nuclear entities even during the 5th post-fertilization cell cycle. Moreover, a considerable proportion of the blastomere nuclei apparently did not show localization of other nucleolar proteins. In conclusion, porcine embryos produced in vitro display a substantial delay in or even lack of the development of functional nucleoli.

Nucleolar proteins and ultrastructure in bovine in vivo developed, in vitro produced, and parthenogenetic cleavage-stage embryos.

In the present study, ribosomal RNA (rRNA) gene activation, monitored through nucleolus development, was studied by autoradiography following (3)H-uridine incubation, transmission electron microscopy, and immunofluorescence confocal laser scanning microscopy of key nucleolar proteins involved in rRNA transcription (topoisomerase I, upstream binding factor, and RNA polymerase I) and processing (fibrillarin, nucleolin, and nucleophosmin) in in vivo developed, in vitro produced, and parthenogenetic bovine embryos. In general, in vivo developed embryos displayed formation of fibrillo-granular nucleoli during the 4th post-fertilization cell cycle. During the previous stages of development, nucleolus precursor bodies (NPBs) were observed. However, on some occasions the initial steps of nucleolus formation were observed already at the 2- and 4-cell stage in cases where such embryos were collected from superovulated animals together with later embryonic stages presenting nucleolar development and autoradiographic labeling. The in vitro produced embryos displayed very synchronous formation of fibrillo-granular nucleoli and autoradiographic labeling during the 4th cell cycle. In vivo developed and in vitro produced embryos displayed allocation of nucleolar proteins to fibrillar and granular compartments of the developing nucleoli during the 4th cell cycle. The parthenogenetic embryos typically displayed formation of fibrillo- granular nucleoli during the 5th cell cycle and autoradiographic labeling was not observed until the morula stage. Moreover, the 1-, 2-, and 4-cell parthenogenetic embryos practically lacked NPBs. On the other hand, parthenogenetic embryos displayed allocation of nucleoar proteins to nuclear entities during the 4th cell cycle. In conclusion, both in vivo developed and in vitro produced bovine embryos displayed activation of transcription and nucleolar development during the 4th cell cycle. However, in vivo developed embryos flushed together with later developmental stages displayed premature activation of these processes. Parthenogenetic bovine embryos, on the other hand, displayed a delayed activation.

Driving current through single organic molecules.

We investigate electronic transport through two types of conjugated molecules. Mechanically controlled break junctions are used to couple thiol end groups of single molecules to two gold electrodes. Current-voltage characteristics ( IVs) of the metal-molecule-metal system are observed. These IVs reproduce the spatial symmetry of the molecules with respect to the direction of current flow. We hereby unambiguously detect an intrinsic property of the molecule and are able to distinguish the influence of both the molecule and the contact to the metal electrodes on the transport properties of the compound system.

Nucleolar protein allocation and ultrastructure in bovine embryos produced by nuclear transfer from granulosa cells.

In the present study immunofluorescence confocal laser scanning microscopy, autoradiography following (3)H-uridine incubation and transmission electron microscopy were used to evaluate the nucleolar protein localization, transcriptional activity, and nucleolar ultrastructure during genomic re-programming in bovine embryos reconstructed by nuclear transfer from granulosa cells into non-activated cytoplasts followed by activation. During the 1st cell cycle (1-cell embryos), no autoradiographic labelling was detected. Ultrastructurally, nucleoli devoid of a granular component were observed. During the 2nd cell cycle (2-cell embryos) autoradiographic labelling was also lacking and the embryos displayed varying degrees of nucleolar inactivation. During both the 3rd (4-cell embryos) and 4th (tentative 8-cell embryos), cell cycles autoradiographic labelling was lacking in some embryos, while others displayed labelling and associated formation of fibrillo-granular nucleoli. During the 5th cell cycle (tentative 16-cell embryos), all embryos displayed autoradiographic labelling and fibrillo-granular nucleoli. In some blastomeres, however, deviant nucleolar ultrastructure was observed. During the first cell cycle labelling of RNA polymerase I, fibrillarin, upstream binding factor (UBF) and nucleolin (C23) was localized to nuclear entities. During the 2nd cell cycle, only labelling of RNA polymerase I and fibrillarin persisted. During the 3rd and 4th cell cycle labelling of fibrillarin persisted, labelling of nucleophosmin (B23) appeared and that of nucleolin re-appeared. During the 5th cell cycle almost all embryos showed complete labelling of all proteins except for UBF, which lacked in more than half of the embryos. In conclusion, bovine granulosa cell nuclear transfer embryos showed re-modelling of the nucleoli to an inactive form followed by re-formation of fibrillo-granular nucleoli. The re-formation of fibrillo-granular nucleoli was initiated already during the 3rd cell cycle, which is one cell cycle earlier than in in vivo- and in vitro-derived bovine embryos. Moreover, in more than half of the embryos, UBF could not be immunocytochemically localized to the nucleolar compartment during the 5th cell cycle indicating lack of developmental potentials.

Nuclear apoptotic changes: an overview.

Apoptosis is a form of active cell death essential for morphogenesis, development, differentiation, and homeostasis of multicellular organisms. The activation of genetically controlled specific pathways that are highly conserved during evolution results in the characteristic morphological features of apoptosis that are mainly evident in the nucleus. These include chromatin condensation, nuclear shrinkage, and the formation of apoptotic bodies. The morphological changes are the result of molecular alterations, such as DNA and RNA cleavage, post-translational modifications of nuclear proteins, and proteolysis of several polypeptides residing in the nucleus. During the last five years our understanding of the process of apoptosis has dramatically increased. However, the mechanisms that lead to apoptotic changes in the nucleus have been only partially clarified. Here, we shall review the most recent findings that may explain why the nucleus displays these striking modifications. Moreover, we shall take into consideration the emerging evidence about apoptotic events as a trigger for the generation of autoantibodies to nuclear components.

Nuclear changes in necrotic HL-60 cells.

Cell death in eukaryotes can occur by either apoptosis or necrosis. Apoptosis is characterized by well-defined nuclear changes which are thought to be the consequence of both proteolysis and DNA fragmentation. On the other hand, the nuclear modifications that occur during necrosis are largely less known. Here, we have investigated whether or not nuclear modifications occur during ethanol-induced necrotic cell death of HL-60 cells. By means of immunofluorescence staining, we demonstrate that the patterns given by antibodies directed against some nuclear proteins (lamin B1, NuMA, topoisomerase IIalpha, SC-35, B23/nucleophosmin) changed in necrotic cells. The changes in the spatial distribution of NuMA strongly resembled those described to occur during apoptosis. On the contrary, the fluorescent pattern characteristic for other nuclear proteins (C23/nucleolin, UBF, fibrillarin, RNA polymerase I) did not change during necrosis. By immunoblotting analysis, we observed that some nuclear proteins (SAF-A, SATB1, NuMA) were cleaved during necrosis, and in the case of SATB1, the apoptotic signature fragment of 70 kDa was also present to the same extent in necrotic samples. Caspase inhibitors did not prevent proteolytic cleavage of the aforementioned polypeptides during necrosis, while they were effective if apoptosis was induced. In contrast, lamin B1 and topoisomerase IIalpha were uncleaved in necrotic cells, whereas they were proteolyzed during apoptosis. Transmission electron microscopy analysis revealed that slight morphological changes were present in the nuclear matrix fraction prepared from necrotic cells. However, these modifications (mainly consisting of a rarefaction of the inner fibrogranular network) were not as striking as those we have previously described in apoptotic HL-60 cells. Taken together, our results indicate that during necrosis marked biochemical and morphological changes do occur at the nuclear level. These alterations are quite distinct from those known to take place during apoptosis. Our results identify additional biochemical and morphological criteria that could be used to discriminate between the two types of cell death. J. Cell. Biochem. Suppl. 36: 19-31, 2001.

Changes in ryanodine receptor-mediated calcium release during skeletal muscle differentiation. II. Resolution of a caffeine-ryanodine paradox.

Our previous study demonstrated a disparity of action between two established pharmacological modulators of the same calcium (Ca2+) release channel, the ryanodine receptor (RyR). Specifically, we observed that caffeine sensitivity was elicited at earlier stages of development than that of ryanodine. In the present study, we offer a hypothesis to resolve this paradox. We provide evidence that ryanodine acts as a pure uncompetitive inhibitor of Ca2+ transport, with respect to Ca2+ itself. This explains why little ryanodine inhibition was observed at low Ca2+ concentrations, while maximal ryanodine inhibition was observed at saturating Ca2+ concentrations. In order to exclude the possibility of nonspecific ryanodine actions as an alternative explanation, we established the phenomenon of capacitative calcium entry (CCE) for L6 cells. Since it is known that CCE is inversely correlated with [Ca2+] of the ER/SR lumen, the extent of CCE is therefore an indirect measure of Ca2+ concentration within the SR. We also demonstrated the functional pathway for Ca2+ entry. Employing pharmacological inhibitors, we found that a T-type plasma membrane channel was predominant in the myoblasts, while an L-type channel was predominant in the adult myotubes. Our data using these inhibitors made nonspecific ryanodine actions an unlikely explanation of the disparity in action between ryanodine and caffeine. Moreover, we found no evidence that inositol trisphosphate, a proposed regulator of CCE for other cells, could influence CCE in L6 cells. We conclude that the disparity between caffeine and ryanodine can be explained by Ca2+ dependence of ryanodine action. This study may also offer an explanation of other studies showing unclear actions of ryanodine binding and action.

Regulation of YKL-40 production by human articular chondrocytes.

OBJECTIVE: YKL-40 (human cartilage glycoprotein 39) is one of the most abundant proteins secreted by cultured chondrocytes. The objectives of the present study were to identify regulators of YKL-40 production in cartilage and chondrocytes and to map the localization of YKL-40 in chondrocytes. METHODS: Human articular chondrocytes and cartilage explants (obtained from subjects at autopsy, from a tissue bank, and from osteoarthritis [OA] patients undergoing total joint replacement surgery) were stimulated with cytokines, growth factors, and other agents. YKL-40 expression was analyzed by Northern blot and polymerase chain reaction. YKL-40 secretion into the media was determined by enzyme-linked immunosorbent assay. RESULTS: YKL-40 production increased to very high levels during the early phase of chondrocyte monolayer culture and in normal cartilage explant cultures as a response to tissue injury. Spontaneous YKL-40 release was higher in OA than in normal cartilage explant cultures. In chondrocyte monolayer cultures, interleukin-1beta (IL-1beta) and transforming growth factor beta (TGFbeta) decreased the levels of secreted YKL-40, and this was associated with a reduction in YKL-40 messenger RNA levels. IL-1beta, but not TGFbeta, reduced YKL-40 production in cartilage explant cultures. Media from explants treated with cycloheximide had no detectable YKL-40, suggesting that the released protein was newly synthesized. Immunofluorescence microscopy showed YKL-40 staining in the Golgi system of the chondrocytes, but YKL-40 could not be detected in the extracellular matrix. CONCLUSION: The spontaneous increase in the production of YKL-40 in the early phase of culture appears to represent a cellular response to changes in the extracellular matrix environment. This, coupled with the profound suppressive effects of IL-1beta and TGFbeta on YKL-40 production, identifies a novel regulatory pattern for this major chondrocyte-derived protein.

A mechanism for the partial insertion of protein kinase C into membranes.

We propose that the principle driving force allowing protein kinase C (PKC) to insert partway into membranes is the transient creation of an interior hydrophilic phase within the membrane. We further suggest that this phase is composed of non-bilayer-forming elements, such as diacylglycerol or phorbol esters. We used the combination of fluorescence resonance energy transfer (using fluorescently labeled phospholipid molecules and the endogenous tryptophan residues of PKC) and fluorescence quenching by the water-soluble reagent potassium iodide. The experimental system used micelles and purified PKC. Our model accounts for both the established kinetic data on PKC as well as the physical requirements of protein-membrane interaction. Moreover, it establishes PKC as the first example of a partially embedded membrane protein, and provides a mechanism to account for its activation.

Nucleolar protein allocation and ultrastructure in bovine embryos produced by nuclear transfer from embryonic cells.

In the present study, immunofluorescence confocal laser scanning microscopy, autoradiography following (3)H-uridine incubation, and transmission electron microscopy were used to evaluate the nucleolar protein localization, transcriptional activity, and nucleolar ultrastructure during genomic reprogramming in bovine embryos reconstructed by nuclear transfer from in vitro-produced bovine morulae to activated cytoplasts. During the first cell cycle (one-cell embryos), no autoradiographic labelling was detected. Ultrastructurally, whorls consisting of densely packed fibrillar material were observed instead of nucleoli. During the second, third, and fourth cell cycle (two-, four-, and tentative eight-cell embryos), autoradiographically unlabelled nuclei contained vacuolated bodies consisting of densely packed fibrillar material. Also, during the fourth cell cycle, the first nucleoplasmic autoradiographic labelling was observed, but still without formation of fibrillo-granular nucleoli. During the fifth cell cycle (tentative 16-cell embryos), the nuclei displayed autoradiographic labelling over both nucleoplasm and presumptive nucleoli, and the formation of fibrillo-granular nucleoli was observed. In a certain proportion of blastomeres, however, abnormal patterns of nucleolar formation and apoptosis were noted. During the first two cell cycles, labelling of RNA polymerase I, fibrillarin, upstream binding factor (UBF), nucleolin (C23), and nucleophosmin (B23) was localized to nuclear entities. During the third cell cycle, labelling of topoisomerase I was observed in addition. During the fourth and fifth cell cycles, a substantial portion of the embryos presented blastomeres that lacked labelling of several of these nucleolar proteins. In conclusion, the nuclear transfer procedure was associated with remodelling of the nucleoli to an inactive form, followed by reformation of fibrillo-granular nucleoli during the fifth cell cycle. Moreover, a certain proportion of blastomeres failed to form functional nucleoli with respect to both ultrastructural organization and protein allocation.

Nucleolar proteins and ultrastructure in preimplantation porcine embryos developed in vivo.

Ribosomal RNA genes are transcribed in the nucleolus. The formation of this organelle after fertilization is essential for embryonic protein synthesis and viability. We have examined nucleolus formation in in vivo-derived porcine embryos by light microscopical autoradiography following 20 min of (3)H-uridine incubation, transmission electron microscopy (TEM), and immunocytochemical localization by confocal laser scanning microscopy of key nucleolar proteins involved in rRNA transcription (nucleolin, upstream binding factor, topoisomerase I, and RNA polymerase I) and processing (fibrillarin, nucleophosmin). During the first two postfertilization cell cycles, TEM revealed fibrillar spheres as the most prominent intranuclear entity of the blastomeres. Fibrillogranular nucleoli were established during the third cell cycle. Initially, fibrillar centers, a dense fibrillar component, and a granular component were formed on the surface of the fibrillar spheres. At the same time, autoradiographic labeling over the nucleoplasm and in particular the nucleoli was detected for the first time. The nucleolar proteins were, in general, not immunocytochemically localized to the presumptive nucleolar compartment until late during the third or early during the fourth cell cycle.

Autoantibodies to DFS 70 kd/transcription coactivator p75 in atopic dermatitis and other conditions.

BACKGROUND: Sera of patients with atopic dermatitis (AD) were found to have autoantibodies that reacted with tissue culture cell substrates in immunohistochemistry to display a characteristic pattern of nuclear distribution of dense fine speckles. The sera also recognized a 70-kd protein on Western immunoblots, and the antigen was termed dense fine speckles 70 kd (DSF70). OBJECTIVE: Because spontaneously occurring autoantibodies could be immune responses to proteins that might be participating in the disease process, it was of interest to identify the antigens driving the autoimmune antibody response. METHODS: A serum containing high-titer antibodies to DFS70 was used to immunoscreen a complementary (c)DNA expression library to isolate cDNA encoding the antigen. After the cDNA was isolated, this was used to express recombinant protein to determine the prevalence of antibody in AD and other conditions. RESULTS: Thirty percent of patients with AD were found to have antibody to recombinant DFS70 in Western immunoblots. Sixteen percent of patients with asthma and 9% of patients with interstitial cystitis had antibodies of the same specificities. The cDNA encoding DFS70 was identical to a transcription coactivator called p75, which had been shown to be required for RNA polymerase II-dependent transcription. Another important finding was that IgE antibodies to DFS70 were also present in AD sera. CONCLUSION: It is suggested that a common basis for the presence of autoantibodies to DFS70 might be related to AD in asthma, interstitial cystitis, and other conditions. A possible role of this antigen-antibody system in pathogenesis remains to be demonstrated, but it appears to be a marker for a subset of patients with AD.

Behavior of nucleolar proteins during the course of apoptosis in camptothecin-treated HL60 cells.

By means of immunofluorescence and immunoelectron microscopy we have studied the fate of different nucleolar components during the apoptotic process in camptothecin-treated HL60 cells. We have found that RNA polymerase I disappeared while UBF was associated with previously described fibrogranular threaded bodies. In contrast, fibrillarin, C23/nucleolin, and B23/nucleophosmin remained detectable in granular material present amid micronuclei of late apoptotic cells. Double immunolabeling experiments showed colocalization of both C23 and B23 with fibrillarin. Immunoblotting analysis showed that UBF was proteolytically degraded, whereas fibrillarin, C23/nucleolin, and B23/nucleophosmin were not. These results may help explain the presence of anti-nucleolar antibodies seen in various pathological disorders.

Activation of ribosomal RNA genes in preimplantation cattle and swine embryos.

Transcription of ribosomal RNA (rRNA) genes occurs in the nucleolus resulting in ribosome synthesis. In cattle and swine embryos, functional ribosome-synthesizing nucleoli become structurally recognizable towards the end of the fourth and third post-fertilization cell cycle, respectively. In cattle, a range of important nucleolar proteins become localized to the nucleolar anlage over several cell cycles and this localization is apparently completed towards the end of the fourth cell cycle. In swine, the localization of these proteins to the anlage is more synchronous and occurs towards the end of the third cell cycle and is apparently completed at the onset of the fourth. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus, serve to evaluate the developmental potential of embryos originating from different embryo technological procedures. By this approach, we have demonstrated that in vitro produced porcine embryos display a lack of localization of nucleolar proteins to the nucleolar anlage as compared with in vivo developed counterparts. Similarly, bovine embryos produced by nuclear transfer from morulae display such deviations as compared with in vitro produced counterparts. Collectively, this information may help to explain the appearance of abnormalities seen in a certain proportion of offspring derived from in vitro produced embryos and after cloning.

New high-DQE imaging plate scanner using the reflected readout laser signal for noise corrections.

Imaging Plates (IPs) are in principle ideal electron detectors combining a large active layer area with a high sensitivity, linear dynamic range detection over 5 orders of magnitude. A moderate resolution and a decreasing detection quantum efficiency (DQE) for higher electron doses limit their use so far. The decrease of the DQE results from linear noise contributed by readout laser instabilities and inhomogeneities of the IP active layer. Here we present data on a new IP drum scanner prototype. This scanner combines twin channel amplification electronics with a new type of readout laser which allows a smaller readout focus and increased stability. The current nominal pixel size is 25 microm, and the measured modulation transfer function (MTF) indicates that further reduction of the scanning step size down to pixel sizes in the range of 12-15 microm should be possible. A unique feature of the new scanner is the simultaneous recording of the reflected readout laser light. The reflected light signal can be used for a posteriori alignment of repeated scans of one individual IP and for a correction of one part of the high spatial frequency noise contribution (reflected light correction). The posteriori alignment now allows an easy conventional gain normalization of the luminescence signal without using special markers on the IP. Both corrections lead to an increase of the DQE for high electron doses.

Nucleolar proteins and nuclear ultrastructure in preimplantation bovine embryos produced in vitro.

The aim of the present investigation was to describe the basic cell biology of the postfertilization activation of rRNA genes using in vitro-produced bovine embryos as a model. We used immunofluorescence confocal laser scanning microscopy and transmission electron microscopy to study nucleolar development in the nuclei of embryos up to the fifth postfertilization cell cycle. During the first cell cycle (1-cell stage), fibrillarin, upstream binding factor (UBF), nucleolin (C23), and RNA polymerase I were localized to distinct foci in the pronuclei, and, ultrastructurally, compact spherical fibrillar masses were the most prominent pronuclear finding. During the second cell cycle (2-cell stage), the findings were similar except for a lack of nucleolin and RNA polymerase I labeling. During the third cell cycle (4-cell stage), fibrillarin, UBF, nucleophosmin, and nucleolin were localized to distinct foci. Ultrastructurally, spherical fibrillar masses that developed a central vacuole over the course of the cell cycle were observed. Early in the fourth cell cycle (8-cell stage), fibrillarin, nucleophosmin, and nucleolin were localized to small bodies that with time developed a central vacuole. UBF and topoisomerase I were localized to clusters of small foci. Ultrastructurally, spherical fibrillar masses with a large eccentric vacuole and later small peripheral vacuoles were seen. Late in the fourth cell cycle, nucleophosmin and nucleolin were localized to large shell-like bodies; and fibrillarin, UBF, topoisomerase I, and RNA polymerase I were localized to clusters of small foci. Ultrastructurally, a presumptive dense fibrillar component (DFC) and fibrillar centers (FCs) were observed peripherally in the vacuolated spherical fibrillar masses. Subsequently, the presumptive granular component (GC) gradually became embedded in the substance of this entity, resulting in the formation of a fibrillo-granular nucleolus. During the fifth cell cycle (16-cell stage), a spherical fibrillo-granular nucleolus developed from the start of the cell cycle. In conclusion, the nucleolar protein compartment in in vitro-produced preimplantation bovine embryos is assembled over several cell cycles. In particular, RNA polymerase I and topoisomerase I are detected for the first time late during the fourth embryonic cell cycle, which coincides with the first recognition of the DFC, FCs, and GC at the ultrastructural level.

Risks of in-vitro production of cattle and swine embryos: aberrations in chromosome numbers, ribosomal RNA gene activation and perinatal physiology.

In cattle, in-vitro production (IVP) of embryos has become a standardized technique; however, increased frequencies of calving problems and larger calves have been reported. In swine, IVP has resulted in only a limited number of piglets. In this paper we present information on cattle and swine embryos produced in vitro by oocyte maturation, fertilization and further embryo culture to the blastocyst stage in vitro. Control in-vivo developed embryos were collected after superovulation. The cattle embryos were processed for fluorescence in-situ hybridization (FISH) with two chromosome-specific probes to detect numerical chromosome aberrations. The swine embryos were processed for transmission electron microscopy and immunocytochemistry with an antibody against RNA polymerase I [essential for ribosomal RNA (rRNA) gene transcription] in order to highlight the post-fertilization development of the nucleolus as a marker for rRNA gene activation. The FISH analyses of the cattle embryos revealed that 72% of IVP blastocysts were mixoploid, i.e. contained both diploid and polyploid cells, versus 25% in vivo. Chromosome abnormalities were observed from the 2-cell stage onwards. The immunocytochemical analyses of the swine embryos revealed that during in-vivo development, RNA polymerase I became localized to multiple foci in the developing nucleoli late during the 4-cell stage. This focal localization of RNA polymerase I was not observed in IVP embryos. In conclusion, IVP embryos may display aberrations in chromosome numbers and rRNA gene activation. The significance of these deviations for fetal and perinatal viability, however, remains unknown. The survival of most calves derived from IVP indicates that a considerable number of these embryos are able to compensate for the adverse effects of the in-vitro procedures.

Nitric oxide production and apoptosis in cells of the meniscus during experimental osteoarthritis.

OBJECTIVE: To examine the pathologic changes in meniscus tissue during experimental osteoarthritis (OA) and to determine the relationship between nitric oxide (NO) synthesis, apoptosis, and meniscus degradation. METHODS: OA was induced in rabbits by anterior cruciate ligament (ACL) transection. Knees were harvested after 9 weeks and assessed for OA severity. Menisci were subjected to histologic, immunohistochemical, and electron microscopic analyses for the presence of nitrotyrosine and apoptosis. Menisci were also cultured for analysis of NO production. RESULTS: All menisci from joints with ACL transection demonstrated degenerative changes. A high number of apoptotic cells was present in the medial part of menisci, which contains chondrocytic cells. Menisci from nonoperated contralateral knees contained only small numbers of cells in apoptosis. Conditioned media from meniscus cultures contained similarly elevated levels of nitrite as cartilage cultures from the same arthritic knees. Nitrotyrosine immunoreactivity, an indicator of in vivo NO production, was prominent in menisci from knees with ACL transection. In addition, menisci from normal knees produced high levels of NO in response to in vitro stimulation with interleukin-1beta or lipopolysaccharide. CONCLUSION: These observations suggest that pathologic changes in menisci are a regular feature of experimentally induced OA and are associated with NO production and meniscus cell apoptosis.

Changes in ryanodine receptor-mediated calcium release during skeletal muscle differentiation.

We have observed a disparity between the actions of caffeine and ryanodine, two agents known to affect the same site of intracellular calcium (Ca2+) release in muscle. The site of intracellular Ca2+ release, the ryanodine receptor (RyR), is established as the route of Ca2+ movement from the sarcoplasmic reticulum (SR) to the cytosol during excitation-contraction coupling. We measured Ca2+ release fluorimetrically in both saponin-permeabilized and intact L6 cells, in response to known modulators (i.e., caffeine and ryanodine), during differentiation in vitro. The undifferentiated L6 cells showed little response to caffeine. However, a substantial caffeine-induced calcium release (caffCR) was evident by Day 3 of differentiation, and was nearly maximal by Day 7 of differentiation. By contrast, ryanodine failed to stimulate Ca2+ release until Day 4, lagging behind the caffeine response. Ryanodine-stimulated Ca2+ release was also maximal by Day 7. Higher concentrations of ryanodine, known to inhibit Ca2+ release, only began to affect caffCR at Day 4, indicating that cells were insensitive to both ryanodine stimulation and ryanodine inhibition prior to this time. Most of the results could be obtained both in permeabilized and intact cells. Using intact cells, we measured the time course of K+ -dependent (i.e., depolarization-induced) Ca2+ release. This time course matched caffeine and not ryanodine-induced Ca2+ release suggesting the action of caffeine was not due to Ca2+ release unrelated to excitation-contraction coupling. These findings suggest that ryanodine binding sites on the RyR may not be functional at early stages of muscle development, that ryanodine sensitivity is a poor indicator of Ca2+ flux through the RyR, or that other proteins are involved in Ca2+ release under certain circumstances.