Low programmed cell death-1 (PD-1) expression in peripheral CD4(+) T cells in Japanese patients with autoimmune type 1 diabetes.

Programmed cell death-1 (PD-1) is a co-stimulatory molecule that inhibits T cell proliferation. We aimed to clarify PD-1 expression in CD4(+) T cells and the association between PD-1 expression and the 7785C/T polymorphism of PDCD1, with a focus on the two subtypes of type 1 diabetes, type 1A diabetes (T1AD) and fulminant type 1 diabetes (FT1D), in the Japanese population. We examined 22 patients with T1AD, 15 with FT1D, 19 with type 2 diabetes (T2D) and 29 healthy control (HC) subjects. Fluorescence-activated cell sorting (FACS) and real-time PCR were utilized to analyse PD-1 expression quantitatively. Genotyping of 7785C/T in PDCD1 was performed using the TaqMan method in a total of 63 subjects (21 with T1AD, 15 with FT1D and 27 HC). FACS revealed a significant reduction in PD-1 expression in CD4(+) T cells in patients with T1AD (mean: 4.2 vs. 6.0% in FT1D, P=0.0450; vs. 5.8% in T2D, P=0.0098; vs. 6.0% in HC, P=0.0018). PD-1 mRNA expression in CD4(+) T cells was also significantly lower in patients with T1AD than in the HC subjects. Of the 63 subjects, PD-1 expression was significantly lower in individuals with the 7785C/C genotype than in those with the C/T and T/T genotypes (mean: 4.1 vs. 5.9%, P=0.0016). Our results indicate that lower PD-1 expression in CD4(+) T-cells might contribute to the development of T1AD through T cell activation.

How psychotropic polypharmacy in schizophrenia begins: a longitudinal perspective.

INTRODUCTION: While patients with schizophrenia are often treated with psychotropic polypharmacy, how and when polypharmacy begins is not well documented. METHODS: A systematic chart review of 300 patients, 100 of whom were psychotropic-free prior to their first visit, was conducted to examine 2-year longitudinal prescription patterns of concomitant psychotropics, in addition to a primary antipsychotic. RESULTS: Overall polypharmacy occurred in 79% patients, with 2-year rates of the use of hypnotics, benzodiazepine derivative anxiolytics, anticholinergic drugs, antidepressants, and mood stabilizers were 56.7, 49.7, 38.3, 21.3, and 14.0%, respectively. Once polypharmacy had started, it was continued until their final visit in >70% of the patients. In a subgroup of 100 psychotropic-free patients, mood stabilizers, antidepressants, anticholinergic antiparkinsonian drugs, anxiolytics, and hypnotics were initiated after 2.3, 2.3, 2.1, 1.6, and 1.5 antipsychotics had been prescribed, respectively (mean duration before the introduction of a concomitant drug in days: 17.7, 121.6, 86.4, 32.1, and, 57.7, respectively). CONCLUSION: Routine practice deviates significantly from algorithms--with polypharmacy often being initiated early, often a without trial of other options, and once started commonly stays.

Small number of HTLV-1-positive cells frequently remains during complete remission after allogeneic hematopoietic stem cell transplantation that are heterogeneous in origin among cases with adult T-cell leukemia/lymphoma.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can provide long-term remission for patients with adult T-cell leukemia/lymphoma (ATLL) caused by human retrovirus, human T-lymphocyte virus (HTLV-1). To understand how HTLV-1-positive cells including ATLL cells were suppressed by allo-HSCT, we examined HTLV-1 provirus load and residual ATLL cells in peripheral blood of transplant recipients using PCR-based tests. We found that the copy number of HTLV-1 genome, called provirus, became very small in number after allo-HSCT; however, in most cases, provirus did not disappear even among long-term survivors. Tumor-specific PCR tests demonstrated that most of HTLV-1-positive cells that remained long after transplantation were not primary ATLL cells but donor-derived HTLV-1-positive cells. We also found a case having very low amount of residual disease in peripheral blood even long after transplantation. There was only one recipient in whom we failed to show the presence of HTLV-1 genome and antibody against HTLV-1 even with an extensive search, which strongly suggested the elimination of HTLV-1 after allo-HSCT. These results demonstrated that after allo-HSCT the small amount of residual HTLV-1-positive cells were heterogeneous in origin and that long-term disease control for ATLL could be obtained without the complete elimination of HTLV-1.

Dynamic behavior of microtubules during dynein-dependent nuclear migrations of meiotic prophase in fission yeast.

During meiotic prophase in fission yeast, the nucleus migrates back and forth between the two ends of the cell, led by the spindle pole body (SPB). This nuclear oscillation is dependent on astral microtubules radiating from the SPB and a microtubule motor, cytoplasmic dynein. Here we have examined the dynamic behavior of astral microtubules labeled with the green fluorescent protein during meiotic prophase with the use of optical sectioning microscopy. During nuclear migrations, the SPB mostly follows the microtubules that extend toward the cell cortex. SPB migrations start when these microtubules interact with the cortex and stop when they disappear, suggesting that these microtubules drive nuclear migrations. The microtubules that are followed by the SPB often slide along the cortex and are shortened by disassembly at their ends proximal to the cortex. In dynein-mutant cells, where nuclear oscillations are absent, the SPB never migrates by following microtubules, and microtubule assembly/disassembly dynamics is significantly altered. Based on these observations, together with the frequent accumulation of dynein at a cortical site where the directing microtubules interact, we propose a model in which dynein drives nuclear oscillation by mediating cortical microtubule interactions and regulating the dynamics of microtubule disassembly at the cortex.

Circulating levels and bone contents of bone gamma-carboxyglutamic acid-containing protein are decreased in streptozocin-induced diabetes. Possible marker for diabetic osteopenia.

To investigate the pathophysiology of diabetic osteopenia, circulating levels and bone contents of bone gamma-carboxyglutamic acid-containing protein (BGP) were measured in streptozocin-induced diabetic rats . Plasma calcium and total protein were significantly decreased (P less than .01) in the diabetic group, and the plasma level of BGP in diabetic rats was 19.6 +/- 2.8 (mean +/- SE) ng/ml, which is significantly lower than the value of 89.2 +/- 14.0 ng/ml in control rats (P less than .01). Bone contents of calcium and hydroxyproline per femur were significantly decreased in the diabetic group (P less than .01), and the ratios of bone calcium to hydroxyproline were not different. Bone BGP content per femur in the diabetic group was 669 +/- 58 micrograms, which was also significantly lower compared with 1241 +/- 126 micrograms in control rats (P less than .01). The decreased bone content of BGP is consistent with the hypothesis that BGP synthesis is impaired in insulin-deficient diabetes. Because a relationship between plasma levels of BGP and bone turnover has been established, the low plasma BGP value suggests there is a decrease in bone turnover in diabetic rats. Therefore, we postulate that the low bone turnover is one of the pathological features of diabetic osteopenia and is at least partly responsible for the occurrence of this complication in diabetes mellitus.

Valores energéticos e composição bromatológica do resíduo seco de fecularia associado a carboidrases para frangos de corte em fase de crescimento / [Energy value and bromatological composition of dry residue of cassava associated with carbohydrases for grower broilers]

Foram determinados os valores energéticos e a composição bromatológica do resíduo seco de fecularia (RSF) para frangos de corte, na fase de crescimento, utilizando ou não enzimas carboidrases. Os tratamentos foram distribuídos em esquema fatorial 2x4 + ração referência, sendo uma RR sem adição de RSF e quatro tratamentos experimentais com 10%, 20%, 30% e 40% de inclusão do RSF e a suplementação ou não com carboidrases. A composição química encontrada para o RSF, na MN, foi de 89,86% de matéria seca, 0,98% de proteína bruta, 3519kcal kg-1 de energia bruta, 0,19% de extrato etéreo, 27% de fibra em detergente neutro, 19,5% de fibra em detergente ácido, 0,33% de cálcio, 0,43% de fósforo, 0,46% de potássio e 0,12% de magnésio. O uso de carboidrases proporcionou um aumento de 173 e 213kcal kg-1 nos valores de EMA e EMAn, respectivamente, resultando em 1828kcal kg-1 EMA e 1840kcal kg-1 EMAn. Concluiu-se que os maiores níveis de EMA e EMAn foram encontrados para o nível de inclusão médio do RSF de 35% e que a suplementação enzimática pode promover aumento desses parâmetros em até 12% em dietas para frangos de corte na fase de crescimento.(AU)

The energetic values and the bromatological composition of the dry residue of cassava (DRC) were determined for growing broilers with or without carbohydrase enzymes. The treatments were distributed in a 2x4 + reference diet factorial scheme, with one RD without addition of DRC and four experimental treatments with 10, 20, 30 and 40% inclusion levels of RSF and supplementation or not with carbohydrases. The chemical composition found for DRC in natural matter was 89.86% dry matter, 0.98% crude protein, 3519kcal kg-1 gross energy, 0.19% ether extract, 27% neutral detergent fiber, 19.5% of acid detergent fiber, 0.33% of calcium, 0.43% of phosphorus, 0.46% of potassium and 0.12% of magnesium. The use of carbohydrase resulted in an increase of 173 and 213kcal kg-1 in EMA and EMAn values, respectively, resulting in 1828kcal kg-1 EMA and 1840kcal kg-1 EMAn. It was concluded that the highest levels of AME and AMEn were found for the mean inclusion level of the DRC of 35% and that enzymatic supplementation may promote the increase of these parameters by up to 12% in broiler diets in the growth phase.(AU)

Successful treatment with a chimeric anti-CD20 monoclonal antibody (IDEC-C2B8, rituximab) for a patient with relapsed mantle cell lymphoma who developed a human anti-chimeric antibody.

Mantle cell lymphoma (MCL) has a poor prognosis without cure; the median overall survival ranges only from 3 to 4 years irrespective of conventional therapeutic regimens. IDEC-C2B8 (rituximab), a chimeric monoclonal antibody against the B-cell-specific antigen CD20, induces an evaluable clinical response in patients with MCL with mild toxicities. However, the single agent rituximab cannot cure MCL. Due to its low immunogenicity, an antibody against IDEC-C2B8 (human antichimeric antibody [HACA]) has rarely been produced in vivo. We report a patient with relapsed MCL who was successfully treated with IDEC-C2B8 for over a year although she developed HACA 6 months after the initial administration of IDEC-C2B8 in the phase II clinical trial conducted by Zenyaku Kogyo Co. Ltd. We followed the pharmacokinetics of IDEC-C2B8, the serum HACA titer, and the number of B lymphocytes in the peripheral blood in relation to clinical response. The HACA became undetectable soon after subsequent administrations of IDEC-C2B8. When the serum level of IDEC-C2B8 was kept elevated, clinical responses were apparently observed and HACA disappeared during this response period. There were no significant clinical toxicities related to the appearance of HACA. The present findings suggested that IDEC-C2B8 is effective and safe even in patients who have developed HACA.

The effect of 1,25-dihydroxycholecalciferol on the changes of bone gamma-carboxyglutamic acid-containing protein in bone and serum of developing chick.

The effects of 1,25-dihydroxycholecalciferol(1,25-(OH)2-D3) injection into developing chick embryos on bone gamma-carboxyglutamic acid-containing protein (BGP) levels in bone and serum were observed in relation to those on calcium metabolism using chick embryos and chicks aged from 13 days' incubation to 7 days of age. Chick BGP was determined by radioimmunoassay using antiserum to purified chick BGP. The injection of 1,25-(OH)2-D3 into the developing chick embryos of 13 days' incubation resulted in the increases of bone BGP levels at hatching and at 2 days of age. However, the distribution of bone BGP at hatching was not influenced by 1,25-(OH)2-D3. On the other hand, serum BGP levels were significantly increased by 1,25-(OH)2-D3 at 14, 15, 18 days' incubation and at 2 days of age. The early changes of serum BGP levels after 1,25-(OH)2-D3 treatment were correlated with the increases of serum calcium concentrations and the decreases of inorganic phosphorus concentrations. These results suggest that 1,25-(OH)2-D3 may directly or indirectly mediate BGP synthesis or secretion associated with calcium metabolism.

The changes of bone gamma-carboxyglutamic acid-containing protein in bone and serum of developing chick.

The changes of bone gamma-carboxyglutamic acid-containing protein (BGP) levels in bone and serum were studied in relation to those in calcium metabolism using chick embryos and chicks aged from 13 days' incubation to 8 weeks old. Chick BGP was determined by radioimmunoassay using antiserum to purified chick BGP. BGP levels in bone and serum increased significantly at hatching and then decreased until 3-5 days of age. Thereafter, BGP levels in bone and serum increased gradually until 8 weeks of age. These changes of BGP levels were well correlated with those of serum calcium and inorganic phosphorus concentrations, serum alkaline phosphatase activity and bone calcium content. The molecular size of increased serum BGP at hatching was not different from that of bone BGP. These results suggest that BGP plays a role in hatching and bone formation during chick development.

Regulatory effect of calcium-regulating hormones on the synthesis and/or secretion of bone gamma-carboxyglutamic acid-containing protein in chick embryonic calvaria in vitro.

Regulatory effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3), 24,25-dihydroxyvitamin D3 (24,25-(OH)2-D3), parathyroid hormone (PTH) and calcitonin (CT) on BGP synthesis and/or secretion were studied using chick embryonic calvaria in vitro. BGP contents in calvaria and culture medium were determined by radioimmunoassay using antiserum to purified chick BGP. After 72 h culture, 1,25-(OH)2-D3 (10(-10)-10(-7) M) increased the BGP content in culture medium significantly, and the effect was maximum at 10(-8)M, while the BGP content in calvaria was not changed by 1,25-(OH)2-D3. 24,25-(OH)2-D3 increased the BGP content both in medium at 10(-6) M-10(-5) M after 24 h culture and in calvaria at 5 X 10(-7)-10(-5) M after 48 h culture. PTH increased BGP contents both in calvaria after 72 h culture and medium at 1 U/ml after 24 h culture, while it decreased them at 5-10 U/ml after 24 h culture. CT (0.5-10 U/ml) had no effect on BGP contents in calvaria and medium. 1,25-(OH)2-D3-induced increase of BGP in culture medium was observed from 24 to 72 h of culture and then reached a plateau at 120 h of culture. 1,25-(OH)2-D3-induced increase in the BGP level in medium after 72 h culture was 3-4 times the control value. On the other hand, the BGP content in calvaria was not affected by 1,25-(OH)2-D3 until 72 h of culture and then significantly increased at 120 h. The addition of 1,25-(OH)2-D3 (10(-8) M), 24,25-(OH)2-D3 (10(-6) M), and 1 U PTH (and 1 U CT added to those hormones) into culture medium of chick embryonic calvaria significantly increased BGP contents in calvaria and medium at 72 h after culture. The effect of three hormones was not synergistic, but had a tendency to be greater than that of a single addition of 1,25-(OH)2-D3, 24,25-(OH)2-D3 or PTH. Further addition of 1 U CT into culture medium did not affect the combined effects of 1,25-(OH)2-D3, 24,25-(OH)2-D3 and PTH except for the calcium content in calvaria. The addition of individual calcium-regulating hormones into culture medium of chick embryonic calvaria did not affect the calcium and phosphorus contents in calvaria, but the calcium content in calvaria was significantly increased by the addition of 1,25-(OH)2-D3, 24,25-(OH)2-D3 and PTH to the culture medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of vitamin D3 metabolites in ovariectomized rats.

The effects of two vitamin D3 metabolites, 24R,25-dihydroxyvitamin D3 and 1 alpha,25-dihydroxyvitamin D3, were investigated in ovariectomized rats. The amount of ash in the femur on a defatted dry weight basis was significantly greater in rats treated with 1 microgram/kg 24R,25-dihydroxyvitamin D3, or 0.01 or 0.1 microgram/kg 1 alpha,25-dihydroxyvitamin D3 than in the controls. The concentration of bone gla protein in serum and amounts in the femur were significantly greater in rats treated with 1 or 10 micrograms/kg 24R,25-dihydroxyvitamin D3, but not those given 1 alpha,25-dihydroxyvitamin D3 compared with the controls. These results suggest that 24R,25-dihydroxyvitamin D3 increased bone mass probably through the stimulation of bone formation.

Frequent expression of the c-kit proto-oncogene in canine malignant mammary tumor.

The mammary tumor is one of the popular neoplastic diseases in female dogs. In the present study, the expression of canine c-kit proto-oncogene in mammary tumor specimens was investigated to evaluate its potential usefulness as a tumor marker. By comparing the homology among the nucleotide sequences reported for human mouse, rat and feline c-kit c-DNA, a pair of primers was synthesized for the reverse transcriptase-polymerase chain reaction (RT-PCR) method. The RT-PCR product of canine spleen total RNA was shown to have 756 bp in size and to be highly homologous to the corresponding sequences reported for the mammalian species. The expression of c-kit transcript was detected in 11 mammary tumors of different histopathology including adenocarcinomas, benign and malignant mixed tumors. The level of the transcription in adenocarcinomas was significantly higher than those in malignant mixed tumors. Fifteen canine tumor specimens originated from various tissues were also tested for their c-kit transcript. In all of the mastocytoma samples examined, high expression of the mRNA was detected. Of other 12 tumors, only low level of RT-PCR products were detected in 5 samples, whereas no apparent amplification was observed in 7 tumors. These results indicate that the high expression of c-kit transcript is helpful for the diagnosis of canine mammary tumors.

[Morphological study on biopsied skin materials of Lafora disease].

Skin biopsy of a patient with Lafora disease was performed. The specimen showed numerous PAS-positive materials in the eccrine glands as proposed by Carpenter and Karpati. The patient was a 25-year-old man, and his two brothers and sister were diagnosed as Lafora disease by clinical and autopsy findings. The patient has shown typical clinical course of Lafora disease. Light microscopic findings of the skin revealed 5-7 microns oval or round PAS-positive materials in most of the glandular cells in eccrine glands. Several histochemical stainings were applied to the specimens so that the storage materials in the glandular cells were considered as glucose-polymer (polyglucosan). Electronmicroscopic observation showed the materials adjacent to the nucleus without limiting membrane. The materials were composed of numerous glycogen-like particles associated with a small number of fine filaments and vesicles. It have been not clarified why these materials are present in the sweat glands in Lafora disease. Recently skin biopsies have been underwent in patients of storage disease involving the central nervous system. Especially it is much useful for diagnosis of the disease with no reliable enzymatic assays. Pathogenesis of Lafora disease is still unknown. Therefore, skin biopsy might be convenient to differentiate from other types of myoclonus epilepsy.

[Effect of aging on bone mineral content. Part VII. The evaluation of osteoporosis treatment by the measurement of total body bone mineral content using dual photon absorptiometry].

In order to evaluate the degree of accuracy of measurement of total body bone mineral content (TBBMC) by dual photon absorptiometry (DPA), the TBBMC in four healthy male volunteers were measured serially for 3 to 12 months. In three to six determinations of TBBMC in various stage of radiation source (153-Gd), the coefficients of variation in four subjects were 1.59, 0.74, 1.25 and 1.27%. Thus, the mean CV was 1.22 +/- 0.35% (mean +/- SD). This indicates that the measurement of TBBMC using DPA is an accurate tool for long-term follow up of bone mineral content and up to 1.6% change of TBBMC might be considered to be a significant change in TBBMC. No apparent drift of TBBMC associated with source decay was noticed in the present study. Subsequently, fifteen females with osteoporosis were studied to evaluate the efficacy of certain therapeutic modes. The patients were divided into two groups. Group 1 (n = 10) given 10 to 40 U of elcatonin (eel calcitonin derivative) intramuscularly every week for 3 to 6 months. Group 2 (n = 5) were treated with 0.5 mu/day of oral 1-alpha-OHD3 for 3 to 6 months. The TBBMC of these fifteen patients were followed by DPA (Lunar DP-4). Seven patients out of ten treated with elcatonin (70%) showed significant (up to 1.6% change in TBBMC compared with baseline) increase in TBBMC after 3 to 6 months treatment. The mean percentage change in TBBMC in group 1 was 101.9 +/- 2.7% (mean +/- SD) when the initial TBBMC was taken as 100%.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of myeloperoxidase enhances the chemosensitivity of leukemia cells through the generation of reactive oxygen species and the nitration of protein.

Myeloperoxidase (MPO), a pivotal lineage marker for acute myeloid leukemia (AML), has been also shown to have a prognostic value: a high percentage of MPO-positive blasts correlates to favorable prognosis. To understand the relationship between the expression of MPO in leukemia cells and the response to chemotherapeutic agents, we established MPO-expressing K562 leukemia cell lines and then treated them with cytosine arabinocide (AraC). Cells expressing wild-type MPO, but not mutant MPO that could not mature, died earlier of apoptosis than control K562 cells. Reactive oxygen species (ROS) were generated more in leukemia cells expressing MPO, and the generation was abrogated by MPO inhibitors or antioxidants. Tyrosine nitration of cellular protein also increased more in MPO-expressing K562 cells than control cells after treatment with AraC. In clinical samples, CD34-positive AML cells from high-MPO cases showed a tendency to be sensitive to AraC in the colony-formation assay, and the generation of ROS and the nitration of protein were observed only when the percentage of MPO-expressing cells was high. These data suggest that MPO enhances the chemosensitivity of AML through the generation of ROS and the nitration of proteins.

Bone gamma-carboxyglutamic acid containing protein in the perinatal period.

We measured bone gamma-carboxyglutamic acid-containing protein (BGP), calcium (Ca), phosphorus (P), and alkaline phosphatase (Al-P) in paired maternal and cord sera, and urinary gamma-carboxyglutamic acid (gamma-Gla) in neonates. The circulating BGP was 41.21 +/- 2.47 ng/ml and 7.44 +/- 0.87 ng/ml in the cord (n = 15) and the maternal (n = 14) sera, respectively. The urinary gamma-Gla in the neonates was 147.68 +/- 10.75 mumol/g creatinine (n = 15). The cord serum BGP was significantly higher than the normal adult level. The maternal serum BGP was at the same level as in other adults. It is conceivable that the fetus may produce BGP during gestation, as the cord serum BGP level was significantly higher than the maternal level and there was no correlation between the cord and maternal serum BGP concentrations. The reason for the elevated circulating BGP level in the cord serum is not known, but increased bone turnover may be a factor. The cord serum BGP may include not only carboxylated but also non-gamma-carboxylated GP because of fetal vitamin K deficiency.

A conserved protein, Nuf2, is implicated in connecting the centromere to the spindle during chromosome segregation: a link between the kinetochore function and the spindle checkpoint.

The centromere is crucial for the proper segregation of chromosomes in all eukaryotic cells. We identified a centromeric protein, Nuf2, which is conserved in fission yeast, human, nematode, and budding yeast. Gene disruption of nuf2+ in the fission yeast Schizosaccharomyces pombe caused defects in chromosome segregation and the spindle checkpoint: the mitotic spindle elongated without segregating the chromosomes, indicating that spindle function was compromised, but that this abnormality did not result in metaphase arrest. Certain nuf2 temperature-sensitive mutations, however, caused metaphase arrest with condensed chromosomes and a short spindle, indicating that, while these mutations caused abnormalities in spindle function, the spindle checkpoint pathway remained intact. Metaphase arrest in these cells was dependent on the spindle checkpoint component Mad2. Interestingly, Nuf2 disappeared from the centromere during meiotic prophase when centromeres lose their connection to the spindle pole body. We propose that Nuf2 acts at the centromere to establish a connection with the spindle for proper chromosome segregation, and that Nuf2 function is also required for the spindle checkpoint.

Establishment of human osteoblastic cells derived from periosteum in culture.

We isolated osteoblastic cells derived from human periosteum and established them in culture. Their growth depended on the presence of ascorbic acid, and the doubling time was 40 to 60 h. The requirement for ascorbic acid was used to high production of collagen. These cells produced mainly type I collagen and only small amounts of type III collagen determined by reducing sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis. The total collagen yield was about 10 mg from 2 X 10(7) cells. The cells could be continuously cultured in alpha-minimum essential medium supplemented with 10% fetal bovine serum for 18 to 40 population doubling levels, depending on the age of the donated periosteum. These cells have the ability to calcify when incubated with 2 mM alpha-glycerophosphate-Na2. Calcification as viewed by the naked eye appeared from Day 15 after treatment. Treatment with the active formed vitamin D3, 1, 25 dihydroxyvitamin D3 enhanced calcification significantly and stimulated osteocalcin production. By electron microscopy, cells with many projections on their surfaces showed well-developed rough endoplasmic reticulum and actinlike fibers, and larger numbers of lysosomes, mitochondria, and secretion granules. Many matrix vesicles, in which minerals were initially localized, and well-banded collagen fibrils were seen in the intercellular spaces. These observations demonstrate typical osteoblastic morphology. The above results indicate that cultured cells from human periostem are osteoblastic cells that have the capacity to differentiate into osteocytes and to deposit calcified minerals in response to 1,25 dihydroxyvitamin D3.

Characterization of fission yeast meiotic mutants based on live observation of meiotic prophase nuclear movement.

We characterized four meiotic mutants of the fission yeast Schizosaccharomyces pombe by live observation of nuclear movement. Nuclei were stained with either the DNA-specific fluorescent dye Hoechst 33342 or jellyfish green fluorescent protein (GFP) fused with the N-terminal portion of DNA polymerase alpha. We first followed nuclear dynamics in wild-type cells to determine the temporal sequence of meiotic events: nuclear fusion in the conjugated zygote is immediately followed by oscillatory nuclear movements that continue for 146 min; then, after coming to rest, the nucleus remains in the center of the cell for 26 min before the first meiotic division. Next we examined nuclear dynamics in four meiotic mutants: mei1 (also called mat2), mei4, dhc1, and taz1. Mei1 and mei4 both arrest during meiotic prophase; our observations, however, show that the timing of mei1 arrest is quite different from that of mei4: the mei1 mutant arrests after nuclear fusion but before starting the oscillatory nuclear movements, while the mei4 mutant arrests after the nucleus has completed the oscillatory movements but before the first meiotic division. We also show examples of the dynamic phenotypes of dhc1 and taz1, both of which complete meiosis but exhibit impaired nuclear movement and reduced frequencies of homologous recombination: the dhc1 mutant exhibits no nuclear movement after nuclear fusion, while the taz1 mutant exhibits severely impaired nuclear movement after nuclear fusion.