Search

The pathogenesis of anti-pituitary-specific transcription factor-1 (PIT-1) hypophysitis was gradually revealed as cases emerged. Our comprehensive analysis, including all reported cases, identified a new instance of anti-PIT-1 hypophysitis postimmune checkpoint inhibitor therapy. All 9 patients exhibited extremely low growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) levels; 2 had a slightly atrophic pituitary gland; 4 had thymoma, and 5 had malignant neoplasms of diffuse large B-cell lymphoma (DLBCL) and other origins. Patients with thymoma showed multiple autoimmune diseases. HLA-A*24:02 and/or A*02:06 were present in six and DR53 in 5 cases analyzed. High anti-PIT-1 antibody titers and ectopic PIT-1 expression in the cytosol and nucleus of the tumor tissues were observed in patients with thymoma or DLBCL, whereas it was exclusively observed in the nuclei of a bladder cancer patient. These findings provide new insights into the pathophysiology of paraneoplastic autoimmune hypophysitis., Competing Interests: Conflict of interest: The authors declare no competing interests relevant to the contents of this article., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Endocrinology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: Luscan-Lumish syndrome (LLS) is characterized by postnatal overgrowth, obesity, Chiari I malformation, seizures, and intellectual disability. SET domain-containing protein 2 (SETD2) is a histone methyltransferase, where mutations in the gene are associated with the development of LLS. However, mechanisms underlying LLS remain unclear., Case Description: A 20-year-old man was referred to our hospital because of tall stature. His body height was 188.2 cm (+3.18 SD) and he showed obesity with a body mass index of 28.4 kg/m2. He exhibited acral overgrowth, jaw malocclusion, and prognathism, but no history of seizures, intellectual disability, or speech delay. Serum growth hormone (GH), insulin-like growth factor 1 (IGF-1), and nadir GH levels after administration of 75 g oral glucose were within normal range. Pituitary magnetic resonance imaging showed no pituitary adenoma, but Chiari I malformation. Whole exome sequencing analysis of the proband revealed a de novo heterozygous germline mutation in SETD2 (c.236T>A, p.L79H). Skin fibroblasts derived from the patient grew faster than those from his father and the control subject. In addition, these cells showed enhanced tyrosine phosphorylation and transcriptional activity of signal transducer and activator of transcription 5b (STAT5b) and increased IGF-1 expression induced by GH., Conclusion: This is a mild case of LLS with a novel mutation in SETD2 without neurological symptoms. LLS should be differentiated in a patient with gigantism without pituitary tumors. Although further investigation is necessary, this is the first study to suggest the involvement of aberrant GH signaling in the development of LLS., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: Germline mutations in fumarate hydratase (FH) gene are known to cause hereditary leiomyomatosis and renal cell carcinoma (HLRCC) and are occasionally accompanied with cutaneous and uterine leiomyoma or cortisol-producing adrenocortical hyperplasia. However, the association between FH mutations and cardiac or adrenocortical tumors has remained unknown. Here, we identified a novel deletion in FH, exhibiting cardiac myxoma and subclinical Cushing syndrome due to adrenocortical tumor., Case Description: A 44-year-old man was referred to our hospital for cardiac and adrenal tumor evaluation. He had a history of multiple painful, dermal papules and nodules diagnosed as cutaneous leiomyoma. The surgically resected cardiac tumor was diagnosed as myxoma. The adrenal tumor was clinically diagnosed as subclinical Cushing syndrome. Laparoscopically resected adrenal tumor was pathologically diagnosed as adrenocortical adenoma harboring unique histological findings similar to primary pigmented nodular adrenocortical disease (PPNAD). DNA analysis revealed a germline deletion in FH c0.737delT (p. Phe225Leufs*31) and loss of heterozygosity (LOH) in cardiac myxoma. As a functional analysis of FH in cardiac myxoma, low FH protein expression with elevated 2-succinocysteine (2SC), a marker of FH dysfunction, was immunohistochemically detected. However, in adrenocortical tumor, LOH of FH was not detected, and FH or 2SC expression was not altered., Conclusions: This is the first case of HLRCC complicated by cardiac myxoma. LOH of FH deletion and its dysfunction were identified in cardiac myxoma. The association between FH deletion and adrenocortical lesion, however, needs to be further clarified., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Basaloid squamous cell carcinoma (BSCC) is a rare and poorly differentiated variant of typical squamous cell carcinoma, and is characterised in part by activation of the Wnt signalling pathway. We previously demonstrated that constitutive activation of the Wnt signalling pathway by epigenetic silencing of secreted frizzled-related protein 4 (SFRP4) is observed in this tumour. Increasing evidence shows that the Wnt signalling pathway cross-talks with other developmental pathways, including the Hedgehog (HH) pathway. The HH pathway is stimulated by inactivating mutations of PTCH1, which have a well-described oncogenic role in basal cell carcinoma (BCC) of the skin. We employed polymerase chain reaction followed by direct sequencing to detect inactivating mutations of PTCH1 using archival tissue samples of 30 oesophageal BSCCs. The frequency of PTCH1 mutation was compared to that of Wnt component genes that we reported previously. We found PTCH1 mutations in 53.3% (16/30) of cases, revealing T1195S as a hotspot mutation. This frequency is quite high for cancers other than BCC of the skin, and PTCH1 mutations were almost mutually exclusive with mutations in APC, Axin1 and Axin2. Considering the fact that activation of Wnt signalling via down-regulation of APC and SFRP5 due to promoter methylation is observed in BCC of the skin, Wnt signalling activation in oesophageal BSCC might be a secondary effect of the PTCH1-inactivating mutations. These findings suggest that the HH and Wnt pathways coordinately contribute to tumourigenesis in oesophageal BSCC. Furthermore, this study provides a potential therapeutic application for HH pathway inhibitors in oesophageal BSCC with highly malignant potential., (© The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: Anti-pituitary-specific transcriptional factor 1 (PIT-1) antibody syndrome is characterized by acquired growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) deficiencies associated with circulating anti-PIT-1 antibodies. Although autoimmunity to PIT-1 has been suggested as a pathogenesis, the precise mechanism of the syndrome remains unclarified., Objective: To elucidate the involvement of antibody- or cell-mediated immunity in anti-PIT-1 antibody syndrome., Materials and Methods: To investigate a direct effect of anti-PIT-1 antibody on pituitary cells, cell proliferation, and cytotoxicity detection assays were performed using patient serum. Enzyme-linked immunospot (ELISpot) assay was performed to evaluate the involvement of PIT-1-reactive cytotoxic T lymphocytes (CTLs). An immunohistochemical analysis using anti-CD4 or anti-CD8 antibody was performed to examine tissue infiltration by CTLs., Results: Patient serum did not exhibit any inhibitory effect on cell proliferation and secretion of GH and PRL in GH3 cells. In addition, complement-dependent cytotoxicity was not detected in patient serum on GH3 cells or primary pituitary cells. The ELISpot assay revealed the presence of CTLs that specifically reacted to the recombinant PIT-1 protein in the patient's peripheral lymphocytes. CD8(+) cell infiltrations, which is the characteristic of CTLs, were observed in the pituitary gland, adrenal gland, stomach, thyroid gland, liver, and pancreas of the patient with anti-PIT-1 antibody syndrome., Conclusions: These results suggest that the anti-PIT-1 antibody is not a cause but a marker of anti-PIT-1 antibody syndrome, in which CTLs play a pivotal role in the pathogenesis.

Iron (Fe) is an essential nutrient in both plants and humans. Fe deficiency on calcareous soil with low Fe availability is a major agricultural problem. Nicotianamine (NA) is one of the Fe chelator in plants, which is involved in metal translocation into seeds, and serves as an antihypertensive substance in humans. In this study, soybean plants overexpressing the barley NA synthase 1 (HvNAS1) gene driven by the constitutive CaMV 35S promoter were produced using Agrobacterium-mediated transformation. The transgenic soybean showed no growth defect and grew normally. The NA content of transgenic soybean seeds was up to four-fold greater than that of non-transgenic (NT) soybean seeds. The level of HvNAS1 expression was positively correlated with the amount of NA, and a high concentration of NA was maintained in the seeds in succeeding generations. The Fe concentration was approximately two-fold greater in transgenic soybean seeds than in NT soybean seeds. Furthermore, the transgenic soybeans showed tolerance to low Fe availability in calcareous soil. Our results suggested that increasing the NA content in soybean seeds by the overexpression of HvNAS1 offers potential benefits for both human health and agricultural productivity.

Objective: The prevalence and clinical characteristics of IgG4-related hypophysitis remain unclear due to the limited number of case reports. Therefore, in this study, we screened consecutive outpatients with hypopituitarism and/or diabetes insipidus (DI) to estimate its prevalence., Methods: A total of 170 consecutive outpatients with hypopituitarism and/or central DI were screened at Kobe University Hospital for detecting IgG4-related hypophysitis by pituitary magnetic resonance imaging, measuring serum IgG4 concentrations, assessing the involvement of other organs, and carrying out an immunohistochemical analysis to detect IgG4-positive cell infiltration., Results: Among the screened cases, 116 cases were excluded due to diagnosis of other causes such as tumors and congenital abnormalities. Additionally, 22 cases with isolated ACTH deficiency were analyzed and were found not to meet the criteria of IgG4-related hypophysitis. The remaining 32 cases were screened and seven were diagnosed with IgG4-related hypophysitis, of which three cases were diagnosed by analyzing pituitary specimens. IgG4-related hypophysitis was detected in 30% (seven of 23 patients) of hypophysitis cases and 4% of all hypopituitarism/DI cases. The mean age at the onset of IgG4-related hypophysitis was 61.8±8.8 years, and the serum IgG4 concentration was 191.1±78.3 mg/dl (normal values 5-105 mg/dl and values in IgG4-related disease (RD) ≥135 mg/dl). Pituitary gland and/or stalk swelling was observed in six patients, and an empty sella was observed in one patient. Multiple co-existing organ involvement was observed in four of the seven patients prior to the onset of IgG4-related hypophysitis., Conclusion: These data suggest that the prevalence of IgG4-related hypophysitis has been underestimated. We should also consider the possibility of the development of hypopituitarism/DI caused by IgG4-related hypophysitis during the clinical course of other IgG4-RDs.

Background: Liver dysfunction in adult hypopituitary patients with GH deficiency (GHD) has been reported and an increased prevalence of nonalcoholic fatty liver disease (NAFLD) has been suggested., Objective: The objective of the present study was to elucidate the pathophysiology of the liver in adult hypopituitary patients with GHD., Patients and Methods: We recruited 69 consecutive Japanese adult hypopituitary patients with GHD and examined the prevalence of NAFLD by ultrasonography and nonalcoholic steatohepatitis (NASH) by liver biopsy. Patients had been given routine replacement therapy except for GH. We compared these patients with healthy age-, gender-, and BMI-matched controls. We further analyzed the effect of GH replacement therapy on liver function, inflammation and fibrotic markers, and histological changes., Results: The prevalence of NAFLD in hypopituitary patients with GHD was significantly higher than in controls (77 vs 12%, P<0.001). Of 16 patients assessed by liver biopsy, 14 (21%) patients were diagnosed with NASH. GH replacement therapy significantly reduced serum liver enzyme concentrations in the patients and improved the histological changes in the liver concomitant with reduction in fibrotic marker concentrations in patients with NASH., Conclusions: Adult hypopituitary patients with GHD demonstrated a high NAFLD prevalence. The effect of GH replacement therapy suggests that the NAFLD is predominantly attributable to GHD.

IGF-I induces skeletal muscle hypertrophy by stimulating protein synthesis and suppressing the protein degradation pathway; the downstream signaling pathways Akt-mammalian target of rapamycin (mTOR)-p70-kDA-S6-kinase (p70S6K), and Forkhead box O1 (FoxO1) play essential roles in this regulation. Reactive oxygen species (ROS) modulate the signaling of various growth factors via redox regulation. However, the role of ROS in IGF-I signaling is not fully understood. In this study, we investigated whether ROS regulate the signaling and biological action of IGF-I in C2C12 myocytes. We found that IGF-I induces ROS in C2C12 myocytes. While treatment with H(2)O(2) significantly enhanced IGF-I-induced phosphorylation of the IGF-I receptor (IGF-IR), IGF-IR phosphorylation was markedly attenuated when cells were treated with antioxidants. The downstream signaling pathway, Akt-mTOR-p70S6K was subsequently down-regulated. Furthermore, the phosphorylation of FoxO1 by IGF-I decreased concomitantly with the restoration of the expression of its target genes, Atrogin-1 and muscle RING finger 1, which are related to muscle atrophy. Nox4 knockdown, which is reportedly to produce ROS in insulin signaling, attenuated IGF-I-induced IGF-IR phosphorylation, indicating that Nox4 is involved in the regulation of IGF-I signaling. Importantly, antioxidant treatments inhibited IGF-I-induced myocyte hypertrophy, demonstrating that ROS are necessary for IGF-I-induced myocyte hypertrophy in vitro. These results indicate that ROS play an essential role in the signaling and biological action of IGF-I in C2C12 myocytes.

A highly sensitive quantitative method for assaying nicotianamine (NA) and 2'-deoxymugineic acid (DMA) using liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) was developed. The amino and hydroxyl groups of NA and DMA were derivatized using 9-fluorenylmethoxycarbonyl chloride. The amounts of NA and DMA in 10 mul of xylem sap from rice cultivated under iron (Fe)-sufficient and Fe-deficient conditions were quantified without concentration. In Fe-sufficient plants, the concentrations of NA and DMA were almost equal to that of Fe. In Fe-deficient plants, the concentration of NA did not change significantly, whereas that of DMA increased markedly.

Recent studies have suggested a link between iron-deficiency anemia and Helicobacter pylori infection. In the current study, strains of H. pylori derived from patients with iron-deficiency anemia showed enhanced Fe ion uptake and Fe ion-dependent rapid growth compared with those from patients with non-iron-deficiency anemia. H. pylori with enhanced Fe ion-uptake ability may be a causative factor for iron-deficiency anemia.

A highly sensitive quantitative method for analyzing nicotianamine (NA) by liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) is reported. Fluorenylmethoxycarbonylation of nicotianamine reduced its polarity and enabled its retention in a reversed-phase column. The adoption of N(epsilon)-nicotyllysine (NL) as an internal standard ensured reliable quantification by giving a linear calibration curve drawn between the NA/NL molar ratios of standard solutions injected and the NA/NL area ratios in mass chromatograms. The high sensitivity of this analytical method allowed us to measure the amount of NA. This analytical method has applications to all research concerning NA.

Zinc (Zn), an essential nutrient in cells, plays a vital role in controlling cellular processes such as growth, development, and differentiation. Although the mechanisms of Zn translocation in rice plants (Oryza sativa) are not fully understood, it has recently received increased interest. OsZIP4 is a Zn transporter that localizes to apical cells. Transgenic rice plants overexpressing the OsZIP4 gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter were produced. The Zn concentration in roots of 35S-OsZIP4 transgenic plants was 10 times higher than in those of vector controls, but it was five times lower in shoots. The Zn concentration in seeds of 35S-OsZIP4 plants was four times lower compared with vector controls. Northern blot analysis and quantitative real-time reverse transcription-PCR revealed transcripts of OsZIP4 expression driven by the CaMV 35S promoter in roots and shoots of 35S-OsZIP4 plants, but levels of endogenous OsZIP4 transcripts were low in roots and high in shoots compared with vector controls. Microarray analysis revealed that the genes expressed in shoots of 35S-OsZIP4 plants coincided with those induced in shoots of Zn-deficient plants. These results indicate that constitutive expression of OsZIP4 changes the Zn distribution within rice plants, and that OsZIP4 is a critical Zn transporter that must be strictly regulated.

Nicotianamine (NA), a metal chelator, is ubiquitous in higher plants. In humans, NA inhibits angiotensin I-converting enzyme (ACE), and consequently reduces high blood pressure. Nicotianamine is synthesized from the trimerization of S-adenosylmethionine (SAM) by NA synthase (NAS). Here, we aimed to produce large amounts of NA fermentatively by introducing the Arabidopsis AtNAS2 gene into Saccharomyces cerevisiae strain SCY4. This strain can accumulate up to 100 times the usual amount of SAM, and this is considered desirable for overproduction of NA. Nicotianamine was produced in the engineered yeast, and the NA level increased with incubation time until the stationary phase. The maximum concentration of intracellular NA obtained was 766+/-33 microg/g wet weight. Successful production of NA in S. cerevisiae should pave the way for industrial production of this novel antihypertensive substrate.

To clarify the molecular mechanism that regulates iron (Fe) acquisition in graminaceous plants, a time-course analysis of gene expression during Fe deficiency stress was conducted using a rice 22K oligo-DNA microarray. Twenty-one genes for proteins that function in gene regulation were induced by Fe deficiency. Of these genes, a putative basic helix-loop-helix (bHLH) transcription factor gene, named OsIRO2, was strongly expressed in both roots and shoots during Fe deficiency stress. The expression of OsIRO2 was induced exclusively by Fe deficiency, and not by deficiencies in other metals. Expression of the barley HvIRO2 gene, which is a homologue of OsIRO2, was also induced by Fe deficiency. An in silico search revealed that IRO2 is highly conserved among graminaceous plants, which include wheat, sorghum, and maize. The cyclic amplification and selection of targets (CASTing) technique revealed that OsIRO2 bound preferentially to the sequence 5'-ACCACGTGGTTTT-3', and the electrophoretic mobility shift assay revealed 5'-CACGTGG-3' as the core sequence for OsIRO2 binding. Sequences similar to the OsIRO2-binding sequence were found upstream of several genes that are involved in Fe acquisition, such as OsNAS1, OsNAS3, OsIRT1, OsFDH, OsAPT1, and IDS3. The core sequence of the OsIRO2-binding sequence occurred more frequently in the upstream regions of Fe deficiency-inducible genes than in the corresponding regions of non-inducible genes. These results suggest that IRO2 is involved in the regulation of gene expression under Fe-deficient conditions.

Zinc (Zn) is an essential element for the normal growth of plants but information is scarce on the mechanisms whereby Zn is transported in rice (Oryza sativa L.) plants. Four distinct genes, OsZIP4, OsZIP5, OsZIP6, and OsZIP7 that exhibit sequence similarity to the rice ferrous ion transporter, OsIRT1, were isolated. Microarray and northern blot analysis revealed that OsZIP4 was highly expressed under conditions of Zn deficiency in roots and shoots. Real-time-PCR revealed that the OsZIP4 transcripts were more abundant than those of OsZIP1 or OsZIP3 in Zn-deficient roots and shoots. OsZIP4 complemented a Zn-uptake-deficient yeast (Saccharomyces cerevisiae) mutant, Deltazrt1,Deltazrt2, indicating that OsZIP4 is a functional transporter of Zn. OsZIP4-synthetic green fluorescent protein (sGFP) fusion protein was transiently expressed in onion epidermal cells localized to the plasma membrane. In situ hybridization analysis revealed that OsZIP4 in Zn-deficient rice was expressed in shoots and roots, especially in phloem cells. Furthermore, OsZIP4 transcripts were detected in the meristem of Zn-deficient roots and shoots. These results suggested that OsZIP4 is a Zn transporter that may be responsible for the translocation of Zn within rice plants.

Nicotianamine, a plant-derived chelator of metals, is produced by the trimerization of S-adenosylmethionine catalyzed by nicotianamine synthase. We established transgenic Arabidopsis and tobacco plants that constitutively overexpress the barley nicotianamine synthase gene. Nicotianamine synthase overexpression resulted in increased biosynthesis of nicotianamine in transgenic plants, which conferred enhanced tolerance of high levels of metals, particularly nickel, to plants. Promoter activities of four nicotianamine synthase genes in Arabidopsis were all increased in response to excess nickel, suggesting that nicotianamine plays an important role in the detoxification of nickel in plants. Furthermore, transgenic tobacco plants with a high level of nicotianamine grew well in a nickel-enriched serpentine soil without developing any symptoms of nickel toxicity. Our results indicate that nicotianamine plays a critical role in metal detoxification, and this can be a powerful tool for use in phytoremediation.

Rice plants (Oryza sativa L.) utilize the iron chelators known as mugineic acid family phytosiderophores (MAs) to acquire iron from the rhizosphere. Synthesis of MAs and uptake of MA-chelated iron are strongly induced under conditions of iron deficiency. Microarray analysis was used to characterize the expression profile of rice in response to iron deficiency at the genomic level. mRNA extracted from iron-deficient or iron-sufficient rice roots or leaves was hybridized to a rice array containing 8987 cDNA clones. An induction ratio of greater than 2.0 in roots was observed for 57 genes, many of which are involved in iron-uptake mechanisms, including every identified or predicted step in the methionine cycle and the biosynthesis of MAs from methionine. Northern analysis confirmed that the expression of genes encoding every step in the methionine cycle is thoroughly induced by iron deficiency in roots, and almost thoroughly induced in leaves. A promoter search revealed that the iron-deficiency-induced genes related to iron uptake possessed sequences homologous to the iron-deficiency-responsive cis-acting elements IDE1 and IDE2 in their promoter regions, at a higher rate than that showing no induction under Fe deficiency. These results suggest that rice genes involved in iron acquisition are co-ordinately regulated by conserved mechanisms in response to iron deficiency, in which IDE-mediated regulation plays a significant role.

Nicotianamine (NA), a chelator of metals, is ubiquitously present in higher plants. Nicotianamine aminotransferase (NAAT) catalyzes the amino group transfer of NA in the biosynthetic pathway of phytosiderophores and is essential for iron acquisition in graminaceous plants. The gene that encodes NAAT from barley was introduced into the nongraminaceous plant tobacco, which produces NA but not phytosiderophores. Transgenic tobacco plants (naat tobacco) that constitutively expressed the NAAT gene had young leaves with interveinal chlorosis and flowers that were abnormally shaped and sterile. Endogenous NA was consumed as a result of NAAT overproduction in naat tobacco. The resulting NA shortage caused disorders in internal metal transport, leading to these abnormal phenotypes. In addition to its role in long-distance metal transport, NA may be involved in the regulation of metal transfer within the cells. These results suggest that a shortage of NA impaired the functions of metal-requiring proteins, including transcription factors.