Comparison of load responsiveness of cartilage T1rho and T2 in porcine knee joints: an experimental loading MRI study.

OBJECTIVE: To compare changes in T1rho and T2 values of the femoral cartilage in porcine knee joints under staged loading and unloading conditions. DESIGN: Sixteen porcine knee joints with intact capsules and surrounding muscle were imaged using a custom-made pressure device and 3.0 T magnetic resonance imaging. Sagittal T1rho and T2 images were obtained for the lateral and medial condyles under the following compression loads: none (Load 0), 140 N (Load 140), 300 N (Load 300), and no compression after decompression (Post-load). The percentage changes of cartilage T1rho and T2 values under each loading condition from those at Load 0 were calculated for weight-bearing overall and eight subdivided regions of interest (ROIs) in both femoral condyles. The actual contact pressure under Load 140 and Load 300 was measured using pressure-sensitive film. RESULTS: For the overall ROI, the mean decreases of T1rho and T2 values were 4.4% and 5.1% under Load 140% and 10.9% and 10.6% under Load 300 in the medial condyle and were 5.2% and 4.0% under Load 140% and 10.6% and 6.0% under Load 300 in the lateral condyle. In the medial condyle, the actual contact pressure correlated highly with percentage changes in T1rho (r = -0.84, P < 0.01) and T2 (r = -0.79, P < 0.01), but those correlations were relatively low in the lateral condyle. CONCLUSION: Although there were side-dependent variations in the correlations with actual pressure, cartilage T1rho and T2 showed similarly sensitive responses to applied load.

Hemodynamic assessment of celiaco-mesenteric anastomosis in patients with pancreaticoduodenal artery aneurysm concomitant with celiac artery occlusion using flow-sensitive four-dimensional magnetic resonance imaging.

OBJECTIVES: Many pancreaticoduodenal artery (PDA) aneurysms are associated with celiac artery (CA) stenosis. The pathogenesis of PDA aneurysm may be associated with hemodynamic changes due to CA stenosis/occlusion. The aim of this study was to assess the hemodynamic changes of celiaco-mesenteric anastomosis in patients with PDA aneurysms concomitant with CA occlusion using four-dimensional flow-sensitive magnetic resonance imaging (4D-Flow). METHODS: 4D-Flow was performed preoperatively on five patients. Seven age- and sex-matched individuals were used as controls. Hemodynamic parameters such as flow volume and maximum flow velocity in PDAs, gastroduodenal arteries, common hepatic arteries, and superior mesenteric arteries were compared between both groups. Wall shear stress (WSS) and oscillatory shear index (OSI) were mapped in both groups. RESULTS: In the patient group, 4D-Flow identified retrograde flow of both gastroduodenal arteries and common hepatic arteries. Heterogeneous distribution patterns of both WSS and OSI were identified across the entire PDA in the patient group. OSI mapping showed multiple regions with extremely high OSI values (OSI > 0.3) in all patients. All PDA aneurysms, which were surgically resected, were atherosclerotic. CONCLUSIONS: 4D-Flow identified hemodynamic changes in celiaco-mesenteric arteries in patients with PDA aneurysms with concomitant CA occlusion. These hemodynamic changes may be associated with PDA aneurysm formation.

Bone marrow-derived stromal cells can express neuronal markers by DHA/GPR40 signaling.

The exact origin of neural stem cells in the adult neurogenesis niche remains unknown. Our previous studies, however, indicated an implication of both bone marrow cells as potential progenitors of hippocampal newborn neurons and polyunsaturated fatty acids as ligands of G protein-coupled receptor 40 (GPR40) signaling. Here, we aimed at studying whether bone marrow-derived stromal cells (BMSC) treated by docosahexaenoic acid (DHA) can express neuronal markers in vitro. We focused on implication of DHA/GPR40 signaling for the expression of neural markers in clonally-expanded BMSC from young macaque monkeys. Cell cycle analysis revealed that the DHA plus bFGF treatment induced a decrease of BMSC proliferation and increased the cells in the G0 resting phase. The transitions from nestin-positive progenitors via immature neuronal (beta III-tubulin-positive) to mature neuronal (NF-M and Map2-positive) phenotypes were examined using RT-PCR, Western blot and immunocytochemistry. We detected a significant increase of GPR40 mRNA and protein expression after bFGF induction, being compared with the untreated BMSC. Addition of DHA, a representative GPR40 ligand, led to a significant down-regulation of GPR40, i.e., G protein-coupled receptor-specific internalization, with a subsequent upregulation of neuronal markers such as beta III-tubulin, NF-M and Map2. These data altogether suggest that adult primate BMSC can express neuronal markers with the aid of DHA/GPR40 signaling.

X chromosome inactivation in nuclear transfer ES cells.

Nuclear transfer ES (ntES) cells are established from cloned blastocysts generated by somatic cell nuclear transfer and are expected to be an important resource for regenerative medicine. However, cloned mammals, generated by similar methods, show various abnormalities, which suggest disordered gene regulation. Random X chromosome inactivation (XCI) has been observed to take place in cloned female mouse embryos, but XCI does not necessarily occur according to Xce strength, a genetic element that determines the likelihood of each X chromosome to be inactivated. This observation suggests incomplete reprogramming of epigenetic marks related to XCI. Here, we investigated XCI in ntES cell lines, which were established using differentiated embryoid bodies that originated from a female mouse ES cell line. We examined Xist RNA localization, histone modifications in the Xist locus, and XCI choice. We did not find substantial differences between the ntES lines and their parental ES line. This suggests that the Xist locus and the epigenetic marks involved in XCI are reprogrammed by nuclear transfer and subsequent ntES cell establishment. In contrast to skewed XCI in cloned mice, our observations indicate that normal XCI choice takes place in ntES cells, which supports the goal of safe therapeutic cloning for clinical use.

Establishment of nuclear transfer embryonic stem cell lines from adult somatic cells by nuclear transfer and its application.

Nuclear transfer can be used to generate embryonic stem cell (ntESC) lines from a patient's own somatic cells. We have shown that ntESCs can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. Several reports have already demonstrated that ntESCs can be used in regenerative medicine in order to rescue immunodeficient or infertile phenotypes. However, it is unclear whether ntES cells are identical to fertilized embryonic stem cells (ESCs). This review seeks to describe the phenotype and possible abnormalities of ntESC lines.

Synthesis and antiherpesvirus activities of 5-alkyl-2-thiopyrimidine nucleoside analogues.

Twenty 5-alkyl-2-thiopyrimidine nucleosides were newly synthesized and examined for antiviral activities against herpes simplex virus (HSV), varicella-zoster virus (VZV) and human cytomegalovirus (HCMV). In this study, 2'-deoxy-5-alkyl-2-thiocytidine analogues had lower 50% effective concentration (EC50) values against HSV-1, and 2'-deoxy-5-alkyl-2-thiouridine analogues showed lower EC50 against VZV than their congeners of arabinoside form. Among the compounds examined, 2'-deoxy-5-ethyl and 5-propyl-2-thiocytidine (TN-53 and TN-54) were most potent and selective anti-HSV compounds. Their EC50s were 0.04 and 0.15 microM, and selectivity indexes were more than 7,215 and 1,849, respectively. On the other hand, 2'-deoxy-5-propyl-2-thiouridine (TN-51), 5-bromovinyl-2-thiouracil arabinoside (TN-65) and 5-styryl-2-thiouracil arabinoside (TN-67) were most potent and selective anti-VZV compounds. Their EC50s were 3.1, 3.8 and 2.6 pM for CaQu strain of VZV, respectively, and 2.1 to 3.0 times lower than that of acyclovir. All 2-thiopyrimidine nucleoside analogues did not show antiviral activities against thymidine kinase (TK) negative strains of HSV-1 and VZV. Only three 2-thiocytosine arabinoside compounds showed marginal anti-CMV activities (EC50s were 57-159 pM). All of the five alkyl-2-thio-pyrimidine nucleoside analogues examined were not cytotoxic to human lymphoblastoid cells (RPM18226) and human embryonic fibroblast cells (MRC-5) at 240 microM (100 microg/ml) or more. Regarding the structure-activity relationship of 5-alkyl-2-thiopyrimidine nucleoside analogues, the following remarks will be noted. Elongation of 5-alkyl chain (methyl to ethyl) of 2-thiocytosine in both deoxyribosyl and arabinosyl nucleosides increased anti-HSV-1 activity but not anti-VZV activity. Furthermore, elongation of the same chain (ethyl to propyl) of 2-thiodeoxyuridine increased anti-VZV activity whereas it did not in the case of 2-thiouracil arabinosides.

Transient occurrence of 27 kDa heat-shock protein in the terminal tubule cells during postnatal development of the rat submandibular gland.

It has been suggested that the 27 kDa heat-shock protein (Hsp27) plays a role at crucial cellular checkpoints for proliferation, apoptosis, and differentiation. We examined the immunolocalization of Hsp27 in the rat submandibular gland during postnatal development, wherein acinar cells proliferate and differentiate at earlier postnatal periods. At 2 weeks of age, weak Hsp27 immunoreactivity was distributed diffusely over all gland components. At 3 weeks, Hsp27 immunoreactivity disappeared in most parts of the acini and ducts, but was intensely accumulated in a small cell population located in the acinar center. This population was composed mostly of terminal tubule (TT) type I cells. At 4 weeks, the Hsp27-immunopositive cell population in the acinar center was composed primarily of immature (type II) acinar cells, partly of immature (granulated) intercalated duct (ID) cells, and occasionally of apoptotic cells. After 5 weeks, all acinar components became mature and were no longer immunoreactive for Hsp27. When acinar cell differentiation was accelerated by administration of isoproterenol to 3-week-old rats for 7 days, the number of Hsp27-positive cells was significantly lower than in the control gland at 4 weeks, confirming that Hsp27 expression is downregulated in mature acinar cells. These results suggest that at around 3-4 weeks in postnatal development, the centroacinar TT cells stop proliferating and begin to differentiate into acinar and ID cells, and occasionally undergo apoptosis. Hsp27 is transiently expressed in the centroacinar TT cells during this critical period, and thus may play a role in their differentiation into the immediate descendants.

Enhanced invasiveness of pancreatic adenocarcinoma cells stably transfected with cationic trypsinogen cDNA.

Various studies have described increased expression of cationic trypsinogen in malignant tumor cells. To explore the role of secreted cationic trypsinogen in invasion by cancer cells, we introduced cationic trypsinogen cDNA into Panc-1, a pancreatic adenocarcinoma-derived cell line that lacks expression of endogeneous trypsinogen. Four independent clones (designated Panc-1-Try-7, -9, -11 and -24) stably expressing cationic trypsinogen mRNA were isolated and processed for further study. In a zymographic analysis, gelatinolytic activity for cationic trypsinogen was detectable in serum-free conditioned media obtained from all 4 transfectants but not in media from mock-transfected or parental Panc-1 cells. A Matrigel invasion assay revealed that all trypsinogen-expressing transfectants acquired significantly greater invasive ability than that shown by mock-transfected and parental Panc-1 cells. In addition, enhanced invasiveness of the transfectants was suppressed by FUT-175, a serine protease inhibitor, to the level seen in parental cells. These results provide direct evidence that cationic trypsinogen can increase the invasive ability of carcinoma cells.

Efficient metaphase II transgenesis with different transgene archetypes.

Mammalian genome characterization and biotechnology each require the mobilization of large DNA segments to produce transgenic animals. We recently showed that mouse metaphase II (mII) oocytes could efficiently promote transgenesis (mII transgenesis) when coinjected with sperm and small (<5 kilobases) ubiquitously expressed transgenes (tgs). We have extended this work and now report that mII transgenesis can readily be applied to a range of larger tgs (11.9-170 kilobases), including bacterial and mammalian artificial chromosome (BAC and MAC) constructs. The efficiency of large-construct mII transgenesis was at least as high as that with small constructs; 11-47% of offspring carried the large tgs. More than 95% of these transgenic founders transmitted the tg to offspring. These data demonstrate the ability of mII transgenesis to deliver large tgs efficiently.

Placentomegaly in cloned mouse concepti caused by expansion of the spongiotrophoblast layer.

Hypertrophic placenta, or placentomegaly, has been reported in cloned cattle and mouse concepti, although their placentation processes are quite different from each other. It is therefore tempting to assume that common mechanisms underlie the impact of somatic cell cloning on development of the trophoblast cell lineage that gives rise to the greater part of fetal placenta. To characterize the nature of placentomegaly in cloned mouse concepti, we histologically examined term cloned mouse placentas and assessed expression of a number of genes. A prominent morphological abnormality commonly found among all cloned mouse placentas examined was expansion of the spongiotrophoblast layer, with an increased number of glycogen cells and enlarged spongiotrophoblast cells. Enlargement of trophoblast giant cells and disorganization of the labyrinth layer were also seen. Despite the morphological abnormalities, in situ hybridization analysis of spatiotemporally regulated placenta-specific genes did not reveal any drastic disturbances. Although repression of some imprinted genes was found in Northern hybridization analysis, it was concluded that this was mostly due to the reduced proportion of the labyrinth layer in the entire placenta, not to impaired transcriptional activity. Interestingly, however, cloned mouse fetuses appeared to be smaller than those of litter size-matched controls, suggesting that cloned mouse fetuses were under a latent negative effect on their growth, probably because the placentas are not fully functional. Thus, a major cause of placentomegaly is expansion of the spongiotrophoblast layer, which consequently disturbs the architecture of the layers in the placenta and partially damages its function.

Recruitment of Mec1 and Ddc1 checkpoint proteins to double-strand breaks through distinct mechanisms.

In response to DNA damage, eukaryotic cells activate checkpoint pathways that arrest cell cycle progression and induce the expression of genes required for DNA repair. In budding yeast, the homothallic switching (HO) endonuclease creates a site-specific double-strand break at the mating type (MAT) locus. Continuous HO expression results in the phosphorylation of Rad53, which is dependent on products of the ataxia telangiectasia mutated-related MEC1 gene and other checkpoint genes, including DDC1, RAD9, and RAD24. Chromatin immunoprecipitation experiments revealed that the Ddc1 protein associates with a region near the MAT locus after HO expression. Ddc1 association required Rad24 but not Mec1 or Rad9. Mec1 also associated with a region near the cleavage site after HO expression, but this association is independent of Ddc1, Rad9, and Rad24. Thus, Mec1 and Ddc1 are recruited independently to sites of DNA damage, suggesting the existence of two separate mechanisms involved in recognition of DNA damage.

Comparison of intracytoplasmic sperm injection for inbred and hybrid mice.

We compared the results of intracytoplasmic sperm injection (ICSI) that leads to full term development of hybrid (B6C3F1 and B6D2F1) and inbred (C57BL/6) mouse embryos. Although fertilization and pre-implantation development of C57BL/6 eggs were similar to those of F1 hybrid eggs, post-implantation development of the embryos from C57BL/6 females was significantly poorer than those of the eggs from hybrid females. Reciprocal crosses of C57BL/6 and B6C3F1 gametes revealed that the low rate of post-implantation development of C57BL/6 embryos was due to oocyte factor(s), rather than the sperm factor.

Cloning and characterization of a novel mouse immunoglobulin superfamily gene expressed in early spermatogenic cells.

We cloned and characterized a novel immunoglobulin superfamily gene from the cDNA library of adult mouse testis. This gene was expressed in the spermatogenic cells and hence termed SgIGSF. The predicted SgIGSF protein was composed of 445 amino-acid residues and contained a signal peptide, three extracellular immunoglobulin (Ig)-like domains, a transmembranous domain, and a cytoplasmic domain. SgIGSF mRNA consisted of two size species, 2.1- and 4.5-kb in length. Besides testis, SgIGSF mRNA was also expressed in a variety of organs, including the cerebrum, liver, kidney, and epididymis. The testis and liver expressed both the 2.1- and 4.5-kb transcripts, whereas the cerebrum and epididymis predominantly expressed the 4.5-kb one. In situ hybridization analysis in testis revealed that SgIGSF mRNA signal was localized to the spermatogenic cells from spermatogonia to zygotene spermatocytes. These results suggested that SgIGSF occurs in the plasma membrane of spermatogenic cells during the earlier stages of spermatogenesis.

A variant form of myelodysplastic syndrome with Ph- minor-BCR/ABL transcript.

This study concerns a patient with minor (m)-BCR/ABL transcript-positive and Philadelphia (Ph) chromosome-negative myelodysplastic syndrome (MDS). The patient was a 78-year-old man whose condition was diagnosed as refractory anemia with excess of blasts in transformation. Molecular genetic studies, using reverse transcriptase polymerase chain reaction analysis detected m-BCR/ABL messenger RNA. We used spectral karyotyping to analyze metaphase cells but could not detect a Ph chromosome. Fluorescence in situ hybridization, however, revealed fusion signals of BCR and ABL probes on an apparently normal chromosome 22.

Protein expression and cell organelle behavior in spermatogenic cells.

Spermatogenic cells stage-specifically produce a wide variety of proteins during spermatogenesis, wherein protein expression is coordinated with cell organelle behavior. It has been shown that the Golgi apparatus and the endoplasmic reticulum (ER) are uniquely coordinated with the expression of an immunoglobulin super-family protein, flagellar plasma membrane MC31 (MC31/CE9), and a molecular chaperone, calmegin, respectively. When the Golgi apparatus begins to generate sperm components in the primary spermatocytes, it actively engages in producing proteins for the acrosome in round spermatids and for the flagellum in elongating spermatids. Structurally, the Golgi apparatus is reduced in size during meiotic division, moves from the apical to the basal region (cytoplasmic lobe) when spermatids differentiate from round to elongating phase, and then collapses in the late maturation phase. The ER is distributed uniformly over the entire cytoplasm of spermatocytes and round spermatids, and then moves distally toward the cytoplasmic lobe along the bundles of microtubule, called the manchette, in elongating spermatids. The ER is resorbed into the radial body in late maturation spermatids. MC31/CE9 expresses strong immunostaining twice on the Golgi apparatus during spermatogenesis, first in early pachytene spermatocytes and then in early elongating spermatids. Calmegin expression exactly parallels ER behavior. This mini-review focuses on the unique relationships in spermatogenic cells, particularly those between protein expression and cell organelle behavior.

Hodgkin's disease preceded by unique neurological symptoms.

This is the first case report of Hodgkin's disease (HD) which showed both remission and exacerbation of neurological signs before a confirmed diagnosis of HD. The episodes occurred three times and multiple lesions were involved. Immunoabsorption plasmapheresis and double filtration plasmapheresis were effective for the first episode, whereas, corticosteroids partly improved the second and third episodes. Fever and lymph node swelling were apparent afterward and she was diagnosed as having HD from a supraclavicular lymph node biopsy. The remaining neurologic deficits responded to chemotherapy and radiotherapy. The neurological symptoms were considered as a paraneoplastic syndrome of HD.

DNA methylation variation in cloned mice.

Mammalian cloning has been accomplished in several mammalian species by nuclear transfer. However, the production rate of cloned animals is quite low, and many cloned offspring die or show abnormal symptoms. A possible cause of the low success rate of cloning and abnormal symptoms in many cloned animals is the incomplete reestablishment of DNA methylation after nuclear transfer. We first analyzed tissue-specific methylation patterns in the placenta, skin, and kidney of normal B6D2F1 mice. There were seven spots/CpG islands (0.5% of the total CpG islands detected) methylated differently in the three different tissues examined. In the placenta and skin of two cloned fetuses, a total of four CpG islands were aberrantly methylated or unmethylated. Interestingly, three of these four loci corresponded to the tissue-specific loci in the normal control fetuses. The extent of aberrant methylation of genomic DNA varied between the cloned animals. In cloned animals, aberrant methylation occurred mainly at tissue-specific methylated loci. Individual cloned animals have different methylation aberrations. In other words, cloned animals are by no means perfect copies of the original animals as far as the methylation status of genomic DNA is concerned.

Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei.

Cloning methods are now well described and in almost routine use. However, the frequencies of production of live offspring from activated oocytes remain at < 3% and little is known about the factors that affect these frequencies. The effects of cytokinesis inhibitors, dimethylsulphoxide (DMSO) and the cell cycle of recipient cytoplasm on the cloning of mice were examined. Reconstructed oocytes, which were activated immediately after nucleus injection and cultured without cytochalasin B, developed into blastocysts at a frequency of 30--54% and into live cloned offspring at a frequency of 2--3%. Activated zygotes did not support development to full term after nuclear transfer. Reconstructed oocytes were activated 1--3 h after nuclear transfer and were exposed separately to three inhibitors of cytokinesis (cytochalasin B, cytochalasin D or nocodazole) to examine the toxicity of these inhibitors on cloning. All of the oocytes exposed to nocodazole-containing media formed many small pseudo-pronuclei, whereas with cytochalasin-containing media most of the activated oocytes formed only two pseudo-pronuclei. Despite such differences, 42--61% of reconstructed embryos developed to the morula-blastocyst stage and 1--3% developed to full term in all groups. Addition of 1% (v/v) DMSO to the activation medium significantly improved the frequency of development to the blastocyst stage and full term; however, this improvement did not lead to a higher success rate in the generation of live cloned offspring. These results show that activated mouse oocytes/zygotes are not effective cytoplasmic recipients with the methods described and that the lack of success of cloning is not due to inhibition of cytokinesis.

The role of cyclic AMP response element-binding protein in testosterone-induced differentiation of granular convoluted tubule cells in the rat submandibular gland.

The postnatal development of granular convoluted tubules (GCT) in the duct system of the rodent submandibular gland is known to be androgen-dependent, but the underlying molecular mechanism is unclear. To test the possible role of the transcription factor, cyclic AMP response element-binding protein (CREB), in the androgen-induced differentiation of GCT, the effect of testosterone on the expression and localization of epidermal growth factor (EGF), a marker of GCT cells, and of CREB was examined in the submandibular glands of immature 3-week-old rats. Northern blotting demonstrated increases in both EGF and CREB mRNA 1-4 days after testosterone administration. Immunoprecipitation also indicated that CREB protein was increased in amount with testosterone administration, and that induced CREB was phosphorylated at the serine residue as in the active form of CREB. In situ hybridization demonstrated that cells with CREB mRNA signal first appeared in the distal portions of striated ducts at 1 day and had increased in number by 4 days after giving testosterone, when cells with EGF mRNA signal became evident in the same duct portions. Immunohistochemistry also showed the occurrence of CREB protein in the nuclei of duct epithelial cells before their differentiation into EGF-positive GCT cells. Finally, pieces of submandibular gland from immature rats were cultured in vitro and their expression of EGF mRNA analysed by the reverse transcriptase-polymerase chain reaction. Testosterone in the medium caused a marked enhancement of EGF expression in the gland in 1-4 days, which was attenuated by simultaneous administration of the antisense oligonucleotide for CREB as well as that for the androgen receptor. These results suggest the CREB is upregulated by androgen and has a crucial role in androgen-induced differentiation of GCT in the duct system of the rat submandibular gland.

Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer.

Embryonic stem (ES) cells are fully pluripotent in that they can differentiate into all cell types, including gametes. We have derived 35 ES cell lines via nuclear transfer (ntES cell lines) from adult mouse somatic cells of inbred, hybrid, and mutant strains. ntES cells contributed to an extensive variety of cell types, including dopaminergic and serotonergic neurons in vitro and germ cells in vivo. Cloning by transfer of ntES cell nuclei could result in normal development of fertile adults. These studies demonstrate the full pluripotency of ntES cells.