- [Show abstract] [Hide abstract] ABSTRACT: Semiconductor nanocrystals (Quantum dots, Qdots) have advantages of narrow, symmetric emission spectra with multiple resolvable colors that can be excited simultaneously using a single excitation wavelength. Therefore, different sized Qdots can serve the simultaneous detection of mRNA and protein. The procedure of in situ hybridization (ISH) and immunohistochemistry (IHC) using confocal laser scanning microscopy (CLSM) and Qdots is simpler than combined ISH and IHC using electron microscopy. This method is applicable to the three-dimensional detection of several molecules including mRNA and proteins.
- [Show abstract] [Hide abstract] ABSTRACT: Sick building syndrome (SBS) is a set of several clinically recognizable symptoms reported by occupants of a building without a clear cause. Neuropathy target esterase (NTE) is a membrane bound serine esterase and its reaction with organophosphates (OPs) can lead to OP-induced delayed neuropathy (OPIDN) and nerve axon degeneration. The aim of our study was to determine whether there was a difference in NTE activity in the peripheral blood mononuclear cells (PBMCs) of Japanese patients with SBS and healthy controls and whether PNPLA6 (alias NTE) gene polymorphisms were associated with SBS. We found that the enzymatic activity of NTE was significantly higher (P < 0.0005) in SBS patients compared with controls. Moreover, population with an AA genotype of a single nucleotide polymorphism (SNP), rs480208, in intron 21 of the PNPLA6 gene strongly reduced the activity of NTE. Fifty-eight SNP markers within the PNPLA6 gene were tested for association in a case-control study of 188 affected individuals and 401 age-matched controls. Only one SNP, rs480208, was statistically different in genotype distribution (P = 0.005) and allele frequency (P = 0.006) between the cases and controls (uncorrected for testing multiple SNP sites), but these were not significant by multiple corrections. The findings of the association between the enzymatic activity of NTE and SBS in Japanese show for the first time that NTE activity might be involved with SBS. © 2013 Wiley Periodicals, Inc. Environ Toxicol, 2013.
- [Show abstract] [Hide abstract] ABSTRACT: Background Osteogenesis imperfecta (OI) is a heterogeneous group of inherited disorders that occur owing to the abnormalities in type 1 collagen, and is characterized by increased bone fragility and other extraskeletal manifestations. We report the case of a patient who was diagnosed with OI following subarachnoid hemorrhage (SAH) secondary to a ruptured saccular intracranial aneurysm (IA). Case Presentation A 37-year-old woman was referred to our hospital because of sudden headache and vomiting. She was diagnosed with SAH (World Federation of Neurosurgical Society grade 2) owing to an aneurysm of the middle cerebral artery. She then underwent surgical clipping of the aneurysm successfully. She had blue sclerae, a history of several fractures of the extremities, and a family history of bone fragility and blue sclerae in her son. According to these findings, she was diagnosed with OI type 1. We performed genetic analysis for a single nucleotide G/C polymorphism (SNP) of exon 28 of the gene encoding for alpha-2 polypeptide of collagen 1, which is a potential risk factor for IA. However, this SNP was not detected in this patient or in five normal control subjects. Other genetic analyses did not reveal any mutations of the COL1A1 or COL1A2 gene. The cerebrovascular system is less frequently involved in OI. OI is associated with increased vascular weakness owing to collagen deficiency in and around the blood vessels. SAH secondary to a ruptured IA with OI has been reported in only six cases. Conclusion The patient followed a good clinical course after surgery. It remains controversial whether IAs are caused by OI or IAs are coincidentally complicated with OI.
- [Show abstract] [Hide abstract] ABSTRACT: Skull base metastasis from differentiated thyroid carcinoma including follicular thyroid carcinoma (FTC) and papillary thyroid carcinoma (PTC) is a rare clinical entity. Eighteen FTC cases and 10 PTC cases showing skull base metastasis have been reported. The most common symptom of skull base metastasis from FTC and PTC is cranial nerve dysfunction. Bone destruction and local invasion to the surrounding soft tissues are common on radiological imaging. Skull base metastases can be the initial clinical presentation of FTC and PTC in the presence of silent primary sites. The possibility of skull base metastasis from FTC and PTC should be considered in patients with the clinical symptoms of cranial nerve dysfunction and radiological findings of bone destruction. A variety of genetic alterations in thyroid tumors have been identified to have a fundamental role in their tumorigenesis. Molecular histochemical studies are useful for elucidating the histopathological features of thyroid carcinoma. Recent molecular findings may provide novel molecular-based treatment strategies for thyroid carcinoma.
- [Show abstract] [Hide abstract] ABSTRACT: We modified and tested S/MAR (scaffold/matrix attachment region) episomal vectors. The new vectors would be useful in obtaining cells stably expressing fluorescent-protein tagged transgenes with small, mostly within 10-fold cell-to-cell fluctuations. In the vectors, the same transcript directs episomal replication and expression of transgene/antibiotic marker, and only antibiotic selection without any other extra steps was sufficient to obtain desired stable cells, including those expressing two different proteins simultaneously. Furthermore, the two test cases (i.e., expression of human growth hormone in AtT20 and four PKC isoforms in HEK293) would prove useful in visualizing and analyzing regulatory processes involving these proteins.
- [Show abstract] [Hide abstract] ABSTRACT: There have been several reports of temozolomide (TMZ) treatment of pituitary carcinomas and atypical adenomas. O(6)-methyl-guanine-DNA methyltransferase is not the sole molecule determining the sensitivity to TMZ in pituitary carcinomas and atypical adenomas. The Japan Society of Hypothalamic and Pituitary Tumors study suggests that MSH6, one of mismatch repair pathway enzyme, fulfills a contributory role to the efficacy of TMZ treatment for pituitary carcinomas and atypical adenomas. The preserved MSH6 function might be essential for the responsiveness to TMZ treatment in pituitary carcinomas and atypical adenomas.
- [Show abstract] [Hide abstract] ABSTRACT: In the original manuscript, the word "fluorescein" was erroneously used indistinctly for "fluorescence" and "fluorescent". Furthermore, "cyan fluorescent protein" was misspelled. These errors have been amended in an amended version of the manuscript, which is available from the Molecules website. The authors and publisher apologize for the inconvenience.
- [Show abstract] [Hide abstract] ABSTRACT: Adult growth hormone (GH) deficiency (AGHD) in Japan is diagnosed based on peak GH concentrations during GH provocative tests such as GHRP-2 stimulation test. In this study, we aimed to evaluate the ability of serum insulin-like growth factor-1 (sIGF-1) and urinary GH (uGH) at the time of awakening to diagnose AGHD. Fifty-nine patients with pituitary disease (32 men and 27 women; age 20-85 y (57.5 ± 15.5, mean ± SD) underwent GHRP-2 stimulation and sIGF-1 testing. Thirty-six and 23 patients were diagnosed with and without severe AGHD, respectively based on a peak GH response of <9 ng/mL to GHRP-2 stimulation. Serum IGF-1 was evaluated as a standard deviation score (IGF-1 SDS) based on age and sex. We determined whether uGH levels in urine samples from 42 of the 59 patients at awakening were above or below the sensitivity limit. We evaluated IGF-1 SDS and uGH levels in a control group of 15 healthy volunteers. Values for IGF-1 SDS were significantly lower in patients with, than without (-2.07 ± 1.77 vs.-0.03 ± 0.92, mean ± SD; p < 0.001) AGHD whereas the range of IGF-1 SDS substantially overlapped at >-1.4. IGF-1 SDS discriminated AGHD more effectively in patients aged ≤60 years. The χ(2) test revealed a statistical relationship between uGH and AGHD (test statistic: 7.0104 ≥ χ(2) (1; 0.01) ＝ 6.6349). When IGF-1 SDS is <-1.4 or uGH is below the sensitivity limit, AGHD can be detected with high sensitivity.
- [Show abstract] [Hide abstract] ABSTRACT: In situ hybridization (ISH) at the electron microscopic (EM) level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. EM-ISH is considered to be an important tool for clarifying the intracellular localization of mRNA and the exact site of pituitary hormone synthesis on the rough endoplasmic reticulum. A combined ISH and immunohistochemistry (IHC) under EM (EM-ISH&IHC) approach has sufficient ultrastructural resolution, and provides two-dimensional images of the subcellular localization of pituitary hormone and its mRNA in a pituitary cell. The advantages of semiconductor nanocrystals (quantum dots, Qdots) and confocal laser scanning microscopy (CLSM) enable us to obtain three-dimensional images of the subcellular localization of pituitary hormone and its mRNA. Both EM-ISH&IHC and ISH & IHC using Qdots and CLSM are useful for understanding the relationships between protein and mRNA simultaneously in two or three dimensions. CLSM observation of rab3B and SNARE proteins such as SNAP-25 and syntaxin has revealed that both rab3B and SNARE system proteins play important roles and work together as the exocytotic machinery in anterior pituitary cells. Another important issue is the intracellular transport and secretion of pituitary hormone. We have developed an experimental pituitary cell line, GH3 cell, which has growth hormone (GH) linked to enhanced yellow fluorescein protein (EYFP). This stable GH3 cell secretes GH linked to EYFP upon stimulation by Ca²+ influx or Ca²+ release from storage. This GH3 cell line is useful for the real-time visualization of the intracellular transport and secretion of GH. These three methods from conventional immunohistochemistry and fluorescein imaging allow us to consecutively visualize the process of transcription, translation, transport and secretion of anterior pituitary hormone.
- [Show abstract] [Hide abstract] ABSTRACT: Combined in situ hybridization (ISH) and immunohistochemistry (IHC) under electron microscopy (EM-ISH & IHC) has sufficient ultrastructural resolution to provide two-dimensional images of subcellular localization of pituitary hormone and its mRNA in a pituitary cell. The advantages of semiconductor nanocrystals (Quantum dots; Qdots) and confocal laser scanning microscopy (CLSM) enable us to obtain three-dimensional images of the subcellular localization of pituitary hormone and its mRNA. Both EM-ISH & IHC and ISH & IHC using Qdots and CLSM are useful for understanding the relationship between protein and mRNA simultaneously in two or three dimensions. CLSM observation of rab3B and SNARE proteins such as SNAP-25 and syntaxin revealed that both rab3B and SNARE system proteins play an important role and work together as the exocytotic machinery in anterior pituitary cells. Another important issue is the intracellular transport and secretion of pituitary hormone. An experimental pituitary cell line, the GH3 cell, in which growth hormone (GH) is linked to enhanced yellow fluorescein protein (EYFP), has been developed. This stable GH3 cell secretes GH linked to EYFP upon being stimulated by Ca(2+) influx or Ca(2+) release from storage. This GH3 cell is useful for real-time visualization of the intracellular transport and secretion of GH. These three methods enable us to visualize consecutively the processes of transcription, translation, transport, and secretion of pituitary hormone.
- [Show abstract] [Hide abstract] ABSTRACT: The case presented here describes the clinical evolution of a pituitary carcinoma from an atypical prolactinoma after temozolomide (TMZ) treatment. The mechanism of acquisition of TMZ resistance was analyzed. A 60-year-old woman with atypical prolactinoma had been treated for 7 years with multiple therapies, including dopamine agonists, surgical intervention (5 times), conventional radiotherapy, and radiosurgery. The patient deteriorated as a result of tumor enlargement. Ten cycles of TMZ therapy, 200 mg/m for 5 days every 4 weeks, improved the patient's performance status and caused tumor shrinkage. Six months after discontinuation of TMZ, the tumor progressed into pituitary carcinoma with tumor regrowth and intraventricular dissemination. TMZ therapy was ineffective this time. A sixth surgery and salvage chemotherapy failed to improve the patient's condition, and she died 9 years after the first diagnosis. Throughout the treatment course, O6-methyl-guanine-DNA methyltransferase (MGMT) was immunonegative in the tumor specimens, including the TMZ-refractory pituitary carcinoma. Mutation of p53 was identified in both the atypical prolactinoma and pituitary carcinoma. In contrast, major differences were noted for mismatch repair protein MSH6 immunostaining: Although MSH6 was diffusely immunopositive in the atypical adenoma, it became immunonegative when the tumor evolved into TMZ-refractory pituitary carcinoma. Loss of MSH6 occurred during the progression from an atypical prolactinoma to a pituitary carcinoma, which may have caused resistance to TMZ treatment. This case suggests that preserving MSH6 function is essential for responsiveness to TMZ treatment in MGMT-negative and p53-mutated atypical pituitary adenoma or pituitary carcinoma.
- [Show abstract] [Hide abstract] ABSTRACT: Mouse transgenesis has proven invaluable for analysis of gene function and generation of human disease models. We describe here the development of a pronuclear injection-based targeted transgenesis (PITT) system, involving site-specific integration in fertilized eggs. The system was applied to two different genomic target loci to generate a series of transgenic lines including fluorescent mice, which reproducibly displayed strong, ubiquitous and stable transgene expression. We also demonstrated that knockdown mice could be readily generated by PITT by taking advantage of the reproducible and highly efficient expression system. The PITT system, which circumvents the problem of unpredictable and unstable transgene expression of conventional random-integration transgenic mice, reduces the time, cost and effort needed to generate transgenic mice, and is potentially applicable to both in vivo ‘gain-of-function’ and ‘loss-of-function’ studies.
Dataset: Video S1[Show abstract] [Hide abstract] ABSTRACT: Real-time motion of EGFP-TSHβ granules close to the plasma membrane of the pituitary cells was monitored at 5 s intervals by TIRF microscopy. Scale bar, 20 µm. (0.06 MB AVI)
- [Show abstract] [Hide abstract] ABSTRACT: SIRT1, a NAD-dependent deacetylase, has diverse roles in a variety of organs such as regulation of endocrine function and metabolism. However, it remains to be addressed how it regulates hormone release there. Here, we report that SIRT1 is abundantly expressed in pituitary thyrotropes and regulates thyroid hormone secretion. Manipulation of SIRT1 level revealed that SIRT1 positively regulated the exocytosis of TSH-containing granules. Using LC/MS-based interactomics, phosphatidylinositol-4-phosphate 5-kinase (PIP5K)gamma was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268) in PIP5Kgamma and enhanced PIP5Kgamma enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5Kgamma knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5Kgamma, PI(4,5)P(2), and reduced the secretion of TSH from pituitary cells. These results were also observed in SIRT1-knockout mice. Our findings indicated that the control of TSH release by the SIRT1-PIP5Kgamma pathway is important for regulating the metabolism of the whole body.
Dataset: Video S2[Show abstract] [Hide abstract] ABSTRACT: Real-time motion of EGFP-TSHβ granules close to the plasma membrane of the SIRT1-overexpressing pituitary cells was monitored at 5 s intervals by TIRF microscopy. Scale bar, 20 µm. (0.06 MB AVI)
Dataset: Video S3[Show abstract] [Hide abstract] ABSTRACT: Real-time motion of EGFP-TSHβ granules close to the plasma membrane of SIRT1-RNAi pituitary cells was monitored at 5 s intervals by TIRF microscopy. Scale bar, 20 µm. (0.06 MB AVI)
- [Show abstract] [Hide abstract] ABSTRACT: Sick building syndrome (SBS) is a chronic disorder caused by exposure to diverse indoor environmental or chemical pollutants. This study examined the association between seven detoxification genes (CYP1A1, CYP2E1, EPHX1, GSTM1, GSTT1, GSTP1, and NAT2) and SBS in the Japanese population. One hundred eighty patients with SBS and 401 healthy controls were enrolled in this study. We examined the prevalence for total of eleven genetic polymorphisms of detoxification genes. However, no statistically significant differences in allele and genotype frequency distributions of eleven genetic polymorphisms of these detoxification genes were found between patients and controls. On this basis, we conclude that the polymorphisms that we assessed for the detoxification genes do not contribute to the etiology of SBS.
- [Show abstract] [Hide abstract] ABSTRACT: To investigate the influence of preoperatively elevated growth hormone to bone mass in acromegalic patients, bone mineral density (BMD), and urinary type I collagen N-telopeptide were analyzed in postoperative patients with somatotroph adenomas (SA), in comparison with patients with clinically nonfunctioning adenomas (NF). The mean T and z scores of BMD in the radius of postoperative patients with SA were significantly higher than those of postoperative patients with NF. There was no significant difference in the mean T and z scores of BMD in the lumbar spine between postoperative patients with SA and those with NF. Thus, BMD in the radius is preferentially maintained in postoperative patients with SA. This result suggests that the preoperative elevation of growth hormone and insulin-like growth factor-1 are beneficial in the maintenance of bone mass in acromegalic patients.
- [Show abstract] [Hide abstract] ABSTRACT: Recently, in order to elucidate the role of rab3B in porosome, we have observed the incorporation of rab3B in the secretion of GH through porosome under confocal laser scanning microscopy (CLSM). Transfected cells with GH-EYFP fusion protein and rab3B-ECFP fusion protein were observed under CLSM, which showed the colocalization of EYFP-GH and ECFP-rab3B in the budding configuration of secretory process. These structural and functional images of rab3B imply the incorporation of rab3B in the secretion of GH through porosome.
- [Show abstract] [Hide abstract] ABSTRACT: Combined medical treatment with long acting octreotide and cabergoline is now used with active patients with acromegaly and can reduce serum growth hormone (GH) and insulin-like growth factor-1 levels. In this article, we analyzed again the molecular aspects of the previously reported rare GH-releasing hormone (GHRH)-producing somatotroph adenoma, and introduce the effects of the combined medical treatment using octreotide LAR and cabergoline for GHRH-producing somatotroph adenoma. The present GHRH-producing somatotroph adenoma had a high Ki-67 staining index, weak, and cytoplasmic-dominant immunostaining of somatostatin receptor 2A, and no gsp mutation. This adenoma was predicted to be resistant to the medical therapy using octreotide LAR. In this extremely rare GHRH-producing pituitary somatotroph adenoma, octreotide LAR alone cannot reduce random GH and insulin-like growth factor-1 levels sufficiently, but combined medical therapy with octreotide LAR and cabergoline can reduce them into the normal range. We show that combined medical treatment with octreotide LAR and cabergoline was effective for a somatotroph adenoma that had a high proliferative potential, weak and cytoplasmic-dominant immunostaining of somatostatin receptor 2A, and no gsp mutation, and has active GH production and secretion regulated by locally generated GHRH from adenoma cell.