Functional Analysis of PTH1R Variants Found in Primary Failure of Eruption.

Although many variants of the parathyroid hormone 1 receptor (PTH1R) gene are known to be associated with primary failure of eruption (PFE), the mechanisms underlying the link remains poorly understood. We here performed functional analyses of PTH1R variants reported in PFE patients-namely, 356C>T (P119L), 395C>T (P132L), 439C>T (R147C), and 1148G>A (R383Q)-using HeLa cells with a lentiviral vector-mediated genetic modification. Two particular variants, P119L and P132L, had severe reduction in a level of N-linked glycosylation when compared with wild-type PTH1R, whereas the other 2 showed modest alteration. PTH1R having P119L or P132L showed marked decrease in the affinity to PTH1-34, which likely led to severely impaired cAMP accumulation upon stimulation in cells expressing these mutants, highlighting the importance of these 2 amino acid residues for ligand-mediated proper functioning of PTH1R. To further gain insights into PTH1R functions, we established the induced pluripotent stem cell (iPSC) lines from a patient with PFE and the heterozygous P132L mutation. When differentiated into osteoblastic-lineage cells, PFE-iPSCs showed no abnormality in mineralization. The mRNA expression of RUNX2, SP7, and BGLAP, the osteoblastic differentiation-related genes, and that of PTH1R were augmented in both PFE-iPSC-derived cells and control iPSC-derived cells in the presence of bone morphogenetic protein 2. Also, active vitamin D3 induced the expression of RANKL, a major key factor for osteoclastogenesis, equally in osteoblastic cells derived from control and PFE-iPSCs. In sharp contrast, exposure to PTH1-34 resulted in no induction of RANKL mRNA expression in the cells expressing P132L variant PTH1R, consistent with the idea that a type of heterozygous PTH1R gene mutation would spoil PTH-dependent response in osteoblasts. Collectively, this study demonstrates a link between PFE-associated genetic alteration and causative functional impairment of PTH1R, as well as a utility of iPSC-based disease modeling for future elucidation of pathogenesis in genetic disorders, including PFE.

Molecular characterization of novel reciprocal translocation t(6;14) in an Epstein-Barr virus-transformed B cell precursor.

An in vitro culture of FLEB14 cells, an Epstein-Barr virus-transformed B cell precursor containing the germ line immunoglobulin genes, gave rise to a uniclonally expanded variant, FLEB14 delta 3, which was rearranged at the immunoglobulin heavy-chain gene locus. Cytogenetic analysis showed that FLEB14 delta 3 had a novel reciprocal translocation, t(6;14)(q15;q32). Molecular cloning of the rearranged DNA fragments and determination of their nucleotide sequence revealed that the recombination event was reciprocal, imprecise, and nonhomologous and took place in the S mu region, like those found in Burkitt's lymphoma cells. We propose a molecular model to explain this genetic event which may be relevant to class switch recombination. The translocated sequence of chromosome 6 did not contain any known oncogenes, although the sequence is conserved among mammals. FLEB14 delta 3 did not show tumorigenicity.

Chromosomal localization of human p85 alpha, a subunit of phosphatidylinositol 3-kinase, and its homologue p85 beta.

The human phosphatidylinositol (PI) 3-kinase p85 alpha subunit gene and its homologue p85 beta were assigned to human chromosomes by analysis of their segregation in a panel of somatic cell hybrids using human-specific polymerase chain reaction primers. The p85 alpha locus was only present in hybrids retaining the human chromosome 5q. The presence of the p85 beta locus coincided with the presence of chromosome 19. The precise chromosomal sublocalization of these two genes was then determined by in situ hybridization. We confirmed the localization of the p85 alpha gene at 5q12-q13, as recently described (Cannizzaro, L.A., Skolnik, E.Y., Margolis, B., Croce, C.M., Schlesinger, J. & Huebner, K. (1991). Cancer Res., 51, 3818-3820) and positioned the p85 beta locus at 19q13.2-q13.4.

In vivo competitive studies between normal and common gamma chain-defective bone marrow cells: implications for gene therapy.

Corrective gene transfer into hematopoietic stem cells (HSCs) is being investigated as therapy for X-linked severe combined immunodeficiency (XSCID) and it is hoped that selective advantage of gene-corrected HSCs will help in achieving full immune reconstitution after treatment. Lines of evidence from the results of allogeneic bone marrow transplantation in patients with XSCID support this hypothesis that, however, has not been rigorously tested in an experimental system. We studied the competition kinetics between normal and XSCID bone marrow (BM) cells using a murine bone marrow transplantation (BMT) model. For easy chimerism determination, we used genetic marking with retrovirus-mediated expression of the enhanced green fluorescent protein (EGFP). We found that XSCID BM cells were able to compete with normal BM cells for engraftment of myeloid lineages in a dose-dependent manner, whereas we observed selective repopulation of T, B, and NK cells deriving from normal BM cells. This was true despite the evidence of competitive engraftment of XSCID lineage marker-negative/c-Kit-positive (Lin-/c-Kit+) cells in the bone marrow of treated animals. From these results we extrapolate that genetic correction of XSCID HSCs will result in selective advantage of gene-corrected lymphoid lineages with consequent restoration of lymphocyte populations and high probability of clinical benefit.

Isolation of two members of the rat MAP kinase kinase gene family.

Mitogen-activated protein (MAP) kinase kinase (MAPKK) is a recently characterized activator of MAP kinase (MAPK), and is considered to be regulated by a protooncogene product c-Raf-1. It is, however, unclear whether the signals originating from c-Raf-1 utilize this phosphorylation cascade to lead to oncogenesis. To clarify this point, we isolated rat MAPKK cDNAs, and identified two distinct cDNAs encoding MAPKK and a highly related kinase, both with molecular weights of approximately 45 kDa (MEK1 and MEK2). Genomic Southern blot analyses suggested that MAPKK may form a large gene family.

PDI and glutathione-mediated reduction of the glutathionylated variant of human lysozyme.

A mutant human lysozyme, designated as C77A-a, in which glutathione is bound to Cys95, has been shown to mimic an intermediate in the formation of a disulfide bond during folding of human (h)-lysozyme. Protein disulfide isomerase (PDI), which is believed to catalyze disulfide bond formation and associated protein folding in the endoplasmic reticulum, attacked the glutathionylated h-lysozyme C77A-a to dissociate the glutathione molecule. Structural analyses showed that the protein is folded and that the structure around the disulfide bond, buried in a hydrophobic core, between the protein and the bound glutathione is fairly rigid. Thioredoxin, which has higher reducing activity of protein disulfides than PDI, catalyzed the reduction with lower efficiency. These results strongly suggest that PDI can catalyze the disulfide formation in intermediates with compact structure like the native states in the later step of in vivo protein folding.

Gene therapy for primary immune deficiencies.

Primary immunodeficiency diseases have been important targets of corrective gene transfer approaches since the very early days of gene therapy. The potential for selective survival advantage of gene-corrected cells over populations carrying the mutated, causative gene translates into the possibility of obtaining clinical meaningful results in patients with primary immunodeficiency diseases even if levels of gene transfer are low. This critical prospect has fueled the interest of researchers since the mid-1980s and has recently determined the success of a clinical trial of gene therapy for X-linked severe combined immunodeficiency.

Immunohistochemical study of p53, EGF, EGF-receptor, v-erb B and ras p21 in squamous cell carcinoma of hypopharynx.

We have characterized 24 hypopharyngeal squamous cell carcinomas and 5 normal hypopharyngeal tissues by immunostaining with antibodies against epidermal growth factor (EGF), EGF-Receptor (EGFR), p53, v-erb B, and ras p21. The Avidin-Biotin Complex (ABC) technique was employed. Overexpression of p53 appeared in 17 of 24 cases of squamous cell carcinoma of the hypopharynx (normal mucosa: none, well differentiated: 60%, moderately differentiated: 71.4%, poorly differentiated: 71.4%). Some dysplastic mucosae surrounding cancer lesions showed overexpression of p53. EGF and EGFR tended to be expressed more strongly in carcinoma [EGF: 29.1% (well differentiated: 30%, moderately differentiated: 28.6%, poorly differentiated: 28.6%); EGFR: 50% (well differentiated: 60%, moderately differentiated: 42.9%, poorly differentiated: 42.9%)] than in normal mucosa (EGF: 0%, EGFR: 20%). The v-erb B stained positively in carcinoma [62.5% (well differentiated: 70%, moderately differentiated: 71.4%, poorly differentiated: 42.9%)] but negatively in normal mucosa. These data suggest that genetic mutations of p53 probably play an important role at an early stage of tumorigenesis, and that the networks of EGF, EGFR and v-erb B probably are involved in the development of hypopharyngeal squamous cell carcinoma.

[A history of nursing in Fukuoka Prefecture].

Nurses who had learned the Nightingale's nursing method in the middle of the Meiji Era systematized nursing in Japan. In 1884, modern nursing began in Tokyo. In Kyushu, nursing education started in Fukuoka Hospital ten years later. At that time, the teaching of nurses was done by individual doctors who taught them all the necessary subjects. This study identifies the ethical criteria which the educators of that time had as the goal of nursing education.

[Observations of capillary fluid flow rate by vibration on the small vessel model].

The vibration hazards in peripheral blood vessels as an occupational origin is caused by using pneumatic vibrating tools. In order to know that the major part of causes is whether due to the direct effects of the physical vibration or due to the effects depended on the excitations of peripheral nervous system, an model experiment was employed to estimate the direct effect of the physical vibration. In our experiments, water as fluid was used instead of blood. The silicon capillary tube as same the physical qualities and conditions as blood vessels was vibrated by oscillator. The silicon capillary tube was exposed by following three type of vibration mode to flow axis; i) sine wave of transverse vibration, ii) rectangular wave of transverse vibration, and iii) sine wave of longitudinal vibration. The range of vibration frequency covered 1-2000 Hz. The averaged acceleration by oscillating vibration and the vibration strength level were estimated about 1-200 G and 150 dB, respectively. The observed decreases of water flow rate due to vibration extremely depend on frequency. The decrease ratio of water flow rate by vibration was at maximum 12% down compare with the cases of non vibration. In these observed results by this model experiment, it should be concluded that the direct effect of the physical vibration to the blood flow was not too strong to cause the actual vibration hazards.

Analysis of capillary fluid flow rate by vibration on a small vessel model.

In order to estimate the direct effect of the physical vibration on the peripheral blood flow rate, we measured the water flow rate in a silicon capillary tube whose end was vibrated. Three types of vibration were added to the tube and the frequency of the vibration covered 1-2000 Hz. It is shown that the water flow rate reduces under the vibration and this reduction strongly depends on the frequency and the type of the vibration. The main reason for this dependence is shown to be as follows. The vibration stretches the tube and the average length of the tube is longer under the vibration than when under no vibration, and the average tube radius is smaller under the vibration. This causes the reduction of the flow rate under vibration. Because the stretch of the tube strongly depends on the frequency and the type of the vibration, the above dependence of the water flow rate finally appears.

[A statistical approach to pruritus senilis].

The purpose of this study is to analyse the cause of itching and explore the relationships between pruritus senilis and various stimuli. Sample of 132 men and 238 women with ages ranging from 60 to 96 years were used in our study. Pruritus senilis is not significantly related to cosmetics and soap. Nevertheless, statistically significant correlations were found between pruritus senilis and physical stimuli (e. g., warmth, coldness, dryness). These observations could suggest that pruritus senilis is caused by changes of temperature.

[Epidemiological study on alcohol consumption and physical health in the Kitakyushu area with special reference to industrial structure].

Kitakyushu city is characterized by the following aspects, that is a large amount of alcohol consumption relative to other cities high death rate of liver cirrhosis and chronic liver diseases many recipients of the Daily Life Security a large population of aged people. This study was carried out to investigate the interrelation of industrial structure and drinking status of people living under these situations in Kitakyushu. The close association between drinking and liver diseases was found. Furthermore, a large proportion of drinkers were recipients of the Daily Life Security and this is one of the problems to be resolved. It also became clear that it is not easy to stop drinking for habitual drinkers in spite of the presence of liver diseases and the loss of feeling of satisfaction with life which may be triggered by retirement from job possibly hampers them from problem drinking. With an increase of a population of aged, an increase of alcoholism among them may cause serious, local but social problems. These problems may not be resolved only by the effort of clinical medicine, also macroscopical point of views through social structure, industrial structure, a policy of welfare, public health services etc. are required. A part of the study was presented at the 17th, 18th and 19th Conference of Japanese Medical Society of Alcohol Studies.

[A statistical approach to Yatabe-Guilford personality inventory and manifest anxiety scale of the elderly].

In nursing, if we know an elderly person's mental condition, and that which does not make him/her too tired, this method can be very useful. Using this method we predicted the MAS score from scores of Y-G scales. We obtained the MAS score results by the following formula: MAS score not equal to 0.31 X Lack of objectivity +0.41 X Inferiority feelings -0.15 X Ascendance +0.34 X Y-G Nervousness +0.30 X Y-G Depression +9.54.

Haploinsufficiency of Sf3b1 leads to compromised stem cell function but not to myelodysplasia.

SF3B1 is a core component of the mRNA splicing machinery and frequently mutated in myeloid neoplasms with myelodysplasia, particularly in those characterized by the presence of increased ring sideroblasts. Deregulated RNA splicing is implicated in the pathogenesis of SF3B1-mutated neoplasms, but the exact mechanism by which the SF3B1 mutation is associated with myelodysplasia and the increased ring sideroblasts formation is still unknown. We investigated the functional role of SF3B1 in normal hematopoiesis utilizing Sf3b1 heterozygous-deficient mice. Sf3b1(+/-) mice had a significantly reduced number of hematopoietic stem cells (CD34(-)cKit(+)ScaI(+)Lin(-) cells or CD34(-)KSL cells) compared with Sf3b1(+/+) mice, but hematopoiesis was grossly normal in Sf3b1(+/-) mice. When transplanted competitively with Sf3b1(+/+) bone marrow cells, Sf3b1(+/-) stem cells showed compromised reconstitution capacity in lethally irradiated mice. There was no increase in the number of ring sideroblasts or evidence of myeloid dysplasia in Sf3b1(+/-) mice. These data suggest that SF3B1 plays an important role in the regulation of hematopoietic stem cells, whereas SF3B1 haploinsufficiency itself is not associated with the myelodysplastic syndrome phenotype with ring sideroblasts.