Successful utilization of clozapine for a patient with treatment-resistant schizophrenia after recurrent violent behavior.

BACKGROUND: In patients with schizophrenia, violent behavior is a clinically important factor that prevents their discharge. Clozapine is an effective antipsychotic medication for treatment-resistant schizophrenia, and its usefulness for aggressive behavior has also been suggested. CASE PRESENTATION: We present the case of a 38-year-old male patient diagnosed with schizophrenia who was successfully treated with clozapine after recurrent violent behavior. He was diagnosed with schizophrenia during his adolescence. He was hospitalized for treatment in his teens, but his hallucinations and delusions persisted even after discharge. In his 30s, he became noticeably emotionally unstable, and despite being treated for an adequate period with sufficient doses of several antipsychotics, his symptoms did not improve. This led to repeated hospitalizations triggered by violent behavior toward his parents and siblings within the home. During his fourth hospitalization, clozapine was initiated due to multiple incidents of violence toward nursing staff secondary to hallucinations and delusions. As the dose of clozapine was gradually increased with therapeutic drug monitoring, the patient's hostility, uncooperativeness, and suspiciousness markedly improved, and his aggressive behavior disappeared. He was discharged to a facility on day 194 after starting clozapine and has continued outpatient visits. CONCLUSION: Clozapine was suggested to be effective for aggressive behavior in patients with treatment-resistant schizophrenia and should be actively considered. In such cases, regular measurement of blood concentration is useful for adjusting the dosage of clozapine.

Tubular Endogenous Erythropoietin Protects Renal Function against Ischemic Reperfusion Injury.

Many large-scale studies show that exogenous erythropoietin, erythropoiesis-stimulating agents, lack any renoprotective effects. We investigated the effects of endogenous erythropoietin on renal function in kidney ischemic reperfusion injury (IRI) using the prolyl hydroxylase domain (PHD) inhibitor, Roxadustat (ROX). Four h of hypoxia (7% O2) and 4 h treatment by ROX prior to IRI did not improve renal function. In contrast, 24-72 h pretreatment by ROX significantly improved the decline of renal function caused by IRI. Hypoxia and 4 h ROX increased interstitial cells-derived Epo production by 75- and 6-fold, respectively, before IRI, and worked similarly to exogenous Epo. ROX treatment for 24-72 h increased Epo production during IRI by 9-fold. Immunohistochemistry revealed that 24 h ROX treatment induced Epo production in proximal and distal tubules and worked similarly to endogenous Epo. Our data show that tubular endogenous Epo production induced by 24-72 h ROX treatment results in renoprotection but peritubular exogenous Epo production by interstitial cells induced by hypoxia and 4 h ROX treatment did not. Stimulation of tubular, but not peritubular, Epo production may link to renoprotection.

Single-molecule analysis of intracellular insulin granule behavior and its application to analyzing cytoskeletal dependence and pathophysiological implications.

Introduction: Mobilization of intracellular insulin granules to the plasma membrane plays a crucial role in regulating insulin secretion. However, the regulatory mechanisms of this mobilization process have been poorly understood due to technical limitations. In this study, we propose a convenient approach for assessing intracellular insulin granule behavior based on single-molecule analysis of insulin granule membrane proteins labeled with Quantum dot fluorescent nanocrystals. Methods: This approach allows us to analyze intracellular insulin granule movement with subpixel accuracy at 33 fps. We tracked two insulin granule membrane proteins, phogrin and zinc transporter 8, fused to HaloTag in rat insulinoma INS-1 cells and, by evaluating the tracks with mean-square displacement, demonstrated the characteristic behavior of insulin granules. Results and discussion: Pharmacological perturbations of microtubules and F-actin affected insulin granule behavior on distinct modalities. Specifically, microtubule dynamics and F-actin positively and negatively regulate insulin granule behavior, respectively, presumably by modulating each different behavioral mode. Furthermore, we observed impaired insulin granule behavior and cytoskeletal architecture under chronic treatment of high concentrations of glucose and palmitate. Our approach provides detailed information regarding intracellular insulin granule mobilization and its pathophysiological implications. This study sheds new light on the regulatory mechanisms of intracellular insulin granule mobilization and has important implications for understanding the pathogenesis of diabetes.

Effects of Roxadustat on Erythropoietin Production in the Rat Body.

Anemia is a major complication of chronic renal failure. To treat this anemia, prolylhydroxylase domain enzyme (PHD) inhibitors as well as erythropoiesis-stimulating agents (ESAs) have been used. Although PHD inhibitors rapidly stimulate erythropoietin (Epo) production, the precise sites of Epo production following the administration of these drugs have not been identified. We developed a novel method for the detection of the Epo protein that employs deglycosylation-coupled Western blotting. With protein deglycosylation, tissue Epo contents can be quantified over an extremely wide range. Using this method, we examined the effects of the PHD inhibitor, Roxadustat (ROX), and severe hypoxia on Epo production in various tissues in rats. We observed that ROX increased Epo mRNA expression in both the kidneys and liver. However, Epo protein was detected in the kidneys but not in the liver. Epo protein was also detected in the salivary glands, spleen, epididymis and ovaries. However, both PHD inhibitors (ROX) and severe hypoxia increased the Epo protein abundance only in the kidneys. These data show that, while Epo is produced in many tissues, PHD inhibitors as well as severe hypoxia regulate Epo production only in the kidneys.

Effects of Angiotensin II on Erythropoietin Production in the Kidney and Liver.

The kidney is a main site of erythropoietin production in the body. We developed a new method for the detection of Epo protein by deglycosylation-coupled Western blotting. Detection of deglycosylated Epo enables the examination of small changes in Epo production. Using this method, we investigated the effects of angiotensin II (ATII) on Epo production in the kidney. ATII stimulated the plasma Epo concentration; Epo, HIF2α, and PHD2 mRNA expression in nephron segments in the renal cortex and outer medulla; and Epo protein expression in the renal cortex. In situ hybridization and immunohistochemistry revealed that ATII stimulates Epo mRNA and protein expression not only in proximal tubules but also in collecting ducts, especially in intercalated cells. These data support the regulation of Epo production in the kidney by the renin-angiotensin-aldosterone system (RAS).

Differentiation of endogenous erythropoietin and exogenous ESAs by Western blotting.

Doping tests for the illegal use of erythropoiesis-stimulating agents (ESAs) have been developed. We developed a new Western blotting method to detect and distinguish endogenous erythropoietin (Epo, 35-38 kDa) and exogenous ESAs (epoetin α and ß, 38-42 kDa; darbepoetin α, 47-50 kDa; epoetin ß pegol, 93-110 kDa). Epo and ESAs are glycoproteins and deglycosylation using peptide-N-glycosidase F shifted all Epo and ESA bands except epoetin ß pegol to 22 kDa. We cut the bands of Epo and ESAs from SDS-PAGE gels and analyzed them by Liquid Chromatography/Mass Spectrometry (LC/MS). LC/MS detected all endogenous Epo and exogenous ESAs as deglycosylated 22 kDa Epo, indicating that LC/MS analysis could confirm the presence of Epo or ESA, but could not distinguish between endogenous Epo and exogenous ESAs. We propose the following Epo doping tests: 1) detect Epo or ESAs by Western blotting of the glycosylated form; 2) increase the reliability by the band shift following deglycosylation; and 3) complete confirmation of Epo or ESA by LC/MS analysis using cut gels. One of the advantages of our method is that pre-purification of samples for Epo is not required in our Western blotting.

Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation.

The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG-bound epoetin ß pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Severe hypoxia (7% O2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney but not the liver is the main site of Epo production in control and severe hypoxia. Our method will make the tests for Epo doping and detection easy.

Fludrocortisone stimulates erythropoietin production in the intercalated cells of the collecting ducts.

Erythropoietin has been thought to be secreted to plasma soon after the production because of the difficulty of Western blot analysis and immunohistochemistry. We established the new methods of Western blot analysis and immunohistochemistry. Using the new methods, we investigated the effects of aldosterone and fludrocortisone, an analogue of aldosterone on erythropoietin mRNA and protein production by the kidneys. Aldosterone stimulated Epo and HIF2α mRNA expressions in tubule suspensions and microdissected medullary thick ascending limbs and outer medullary collecting ducts. Western blot analysis showed a recombinant erythropoietin at 34-45 kDa and kidney erythropoietin at 36-40 and 42 kDa, both of which shifted to 22 kDa by deglycosylation. Erythropoietin protein expression was observed in the nephrons but not in the interstitial cells in control condition. Fludrocortisone stimulated erythropoietin mRNA and protein expressions in the distal nephrons, particularly in the intercalated cells of the collecting ducts. These data show that erythropoietin is produced by the nephrons by the regulation of renin-angiotensin-aldosterone system and not by the renal interstitial cells in control condition.

Complete mitochondrial genome of "Xenopus tropicalis" Asashima line (Anura: Pipidae), a possible undescribed species.

The diploid Xenopus tropicalis, with its small nuclear genomic size and short generation time compared to the traditional experimental amphibian X. laevis, is considered a next-generation model animal. Several experimental X. tropicalis lines have been used in research studies. Previous studies showed that the mtDNA sequence of the Asashima line is divergent from other lines and that this line may represent a distinct species. Here, we report the complete nucleotide sequence of this unique X. tropicalis experimental line. The genome is 17,700 bp in length and contains 37 genes commonly found in animal mtDNAs. The 16S rRNA gene sequence in Asashima line differed by over 6% from the standard Nigerian lines (a 3% difference is considered the species threshold in anurans), suggesting that this experimental line is a distinct species from the true X. tropicalis.

Insulin-like factor regulates neural induction through an IGF1 receptor-independent mechanism.

Insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) signalling is required for normal embryonic growth and development. Previous reports indicated that the IGF/IGF1R/MAPK pathway contributes to neural induction and the IGF/IGF1R/PI3K/Akt pathway to eye development. Here, we report the isolation of insulin3 encoding a novel insulin-like ligand involved in neural induction. Insulin3 has a similar structure to pro-insulin and mature IGF ligands, but cannot activate the IGF1 receptor. However, similar to IGFs, Insulin3 induced the gene expression of an anterior neural marker, otx2, and enlarged anterior head structures by inhibiting Wnt signalling. Insulin3 are predominantly localised to the endoplasmic reticulum when otx2 is induced by insulin3. Insulin3 reduced extracellular Wnts and cell surface localised Lrp6. These results suggest that Insulin3 is a novel cell-autonomous inhibitor of Wnt signalling. This study provides the first evidence that an insulin-like factor regulates neural induction through an IGF1R-independent mechanism.

Characterization of the insulin-like growth factor binding protein family in Xenopus tropicalis.

The insulin-like growth factor binding protein (Igfbp) family consists of six members designated Igfbp1-6. Igfbps are involved in many vital biological functions. They physically interact with IGFs (IGF1 and IGF2) and act as carriers, thereby protecting IGFs from proteolytic degradation. Thus, they function as modulators of IGF activity. Furthermore, Igfbps have been reported to have IGF-independent activities. They interact with other proteins, including cell surface proteins, extra-cellular matrix proteins, and potentially intracellular molecules. In Xenopus tropicalis (X. tropicalis), only four igfbp genes (igfbp1, igfbp2, igfbp4, and igfbp5) have been identified, and their expression is not well characterized. We report that X. tropicalis genome lacks the igfbp3 and igfbp6 genes based on synteny analyses. We also examined the spatio-temporal expression patterns of igfbp genes in early X. tropicalis development. Expression analyses indicated that they are differentially expressed during early development. Each igfbp gene showed a characteristic spatial expression pattern. Except for igfbp5, they demonstrated overlapping expression in the pronephros. The Xenopus pronephros is composed of four domains (i.e., the proximal tubule, intermediate tubule, distal tubule, and connecting tubule). Our results showed that at least two igfbp genes are co-expressed in all pronephric domains, suggesting that redundant functions of igfbp genes are required in early pronephric kidney development.