Cas9-mediated DNA cleavage guided by enzymatically prepared 4'-thio-modified RNA.

CRISPR-Cas9-mediated DNA editing relies on guide RNAs (gRNAs) that direct site-specific DNA cleavage by the Cas endonuclease. Because natural gRNA is susceptible to intracellular degradation, it is desirable to chemically protect it for efficient editing. Using 4'-thioribonucleoside 5'-triphosphates and T7 transcription, we have prepared 4'-thio-modified gRNAs that guide Cas9-mediated DNA cleavage. This approach is a simple way to obtain chemically modified RNA suitable for CRISPR-Cas9 DNA editing.

Gene Expression of 4'-Thioguanine DNA via 4'-Thiocytosine RNA.

DNA and RNA nucleotides are ubiquitous molecules that store and transmit genetic information. The emergence of synthetic elements that fulfill the function of DNA and RNA provides an alternative gene expression system. Herein, we demonstrate the gene expression of 4'-thioguanine DNA (dSG DNA) via 4'-thiocytosine RNA (dSC RNA) to give green fluorescent protein (GFPuv) in a single test tube. In replication, transcription, and translation, DNA/RNA polymerases and Escherichia coli (E. coli) ribosome can tolerate the replacement of O4' with S4' in the nucleotide, despite the fact that sulfur has a larger atomic radius than oxygen. Additionally, dSG DNA and dSC RNA acted as alternative genetic polymers to natural DNA and RNA for protein synthesis in artificial cells comprising a reconstituted E. coli gene expression machinery. This work involved simple experiments that are widely used in molecular biology, but which underscore the feasibility of life control by substances other than DNA/RNA nucleotides.

Role of FK506 Binding Protein on Tacrolimus Distribution in Red Blood Cells.

PURPOSE: Tacrolimus is distributed mainly in red blood cells (RBCs) after transfer into blood. This study aimed to evaluate the effect of FK506-binding proteins (FKBPs) on RBC distribution of tacrolimus in a physiological environment. METHODS: Human RBCs were isolated from fresh blood samples from healthy volunteers. The effect of FKBPs on each process of the RBC distribution of tacrolimus was evaluated in vitro. Effect of intracellular FKBPs was assessed by inhibition experiment with rapamycin, which competitively inhibits the binding of tacrolimus to FKBPs. Effect of extracellular FKBPs was examined by pre-exposure of RBCs to FKBP and preincubation of tacrolimus with FKBP. RESULTS: Pretreatment with rapamycin significantly reduced the rate of tacrolimus distribution in RBCs in a concentration-dependent manner. Pre-exposure of RBCs to FKBP12 followed by exposure to tacrolimus significantly decreased tacrolimus distribution in RBCs in a concentration-dependent manner. In addition, preincubation of tacrolimus with FKBP12 significantly reduced the rate of tacrolimus distribution in RBCs. CONCLUSIONS: FKBP played an important role in the distribution of tacrolimus in RBCs. The effect of intracellular and extracellular FKBPs on RBC distribution of tacrolimus in circulating blood was substantial. FKBP was shown as a potential biomarker for predicting the pharmacokinetics and pharmacodynamics of tacrolimus.

Risk for prolonged hospitalization and mortality in aged community acquired pneumonia patients: a retrospective study in Japan.

The present study aimed to reveal; i) risk for prolonged hospitalization and mortality in aged community acquired pneumonia patients, and ii) whether swallowing ability was related to re-hospitalization. The present retrospective study included 92 patients older than 75 years hospitalized with community acquired pneumonia in Takagi Hospital between April 2017 and March 2018. The patients were classified into 3 groups; discharged within 17 days (group I): hospitalized more than 18 days (group II): died during the hospitalization (group III). Swallowing ability was evaluated if available. Univariate analysis indicated males and body mass index (BMI) in group I (n = 24) were higher than group II (n = 46). Group III (n = 22) had low serum albumin, low BMI, and severe disease progression compared with group I. Multivariate analysis demonstrated that group II BMI was lower than group I [odds ratio (OR) = 1.18, p = 0.042]. Group III had lower serum albumin level compared with group I (OR = 81.01, p = 0.025). Diabetes mellitus (p = 0.009), but not swallowing disability, was risk for readmission. Malnutrition represented by low albumin enhanced mortality rate in the pneumonia patients, and low BMI and diabetes mellitus might increase the pneumonia risk.

Ghrelin does not influence cancer progression in a lung adenocarcinoma cell line.

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHSR), is produced in the human stomach. Although ghrelin has therapeutic potential for cancer cachexia, ghrelin treatment may have a concern about accelerating cancer progression. Here, using the human lung adenocarcinoma cell line HLC-1, we investigated the effects of ghrelin on molecular mechanisms linked to cancer progression, including cell viability, proliferation, resistance to apoptosis, and mitochondrial activity. Both types of mouse alveolar epithelial cells (types I and II) expressed the GHSR, as did the human normal airway cell lines BEAS-2B and HLC-1. Treatment with ghrelin (10-2, 10-1, 1, 10 µM) did not affect cell viability or proliferation. Pretreatment of HLC-1 cells with ghrelin (10 µM) did not affect resistance to paclitaxel-induced apoptosis. The parameters of mitochondrial respiration, including basal respiration, proton leak, ATP production, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration, of the HLC-1 cells pretreated with or without ghrelin were unchanged. Taken together, ghrelin does not influence cancer progression in lung adenocarcinoma cells.

The Impacts of Cellular Senescence in Elderly Pneumonia and in Age-Related Lung Diseases That Increase the Risk of Respiratory Infections.

Pneumonia generates considerable negative impacts on the elderly. Despite the widespread uses of vaccines and appropriate antibiotics, the morbidity and mortality of elderly pneumonia are significantly higher compared to the counterparts of young populations. The definitive mechanisms of high vulnerability in the elderly against pathogen threats are unclear. Age-associated, chronic low-grade inflammation augments the susceptibility and severity of pneumonia in the elderly. Cellular senescence, one of the hallmarks of aging, has its own characteristics, cell growth arrest and senescence-associated secretory phenotype (SASP). These properties are beneficial if the sequence of senescence-clearance-regeneration is transient in manner. However, persisting senescent cell accumulation and excessive SASP might induce sustained low-grade inflammation and disruption of normal tissue microenvironments in aged tissue. Emerging evidence indicates that cellular senescence is a key component in the pathogenesis of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), which are known to be age-related and increase the risk of pneumonia. In addition to their structural collapses, COPD and IPF might increase the vulnerability to pathogen insults through SASP. Here, we discuss the current advances in understanding of the impacts of cellular senescence in elderly pneumonia and in these chronic lung disorders that heighten the risk of respiratory infections.

Radiological features and surgical management of lung abscess directly extending into the chest wall: A case report.

INTRODUCTION AND IMPORTANCE: We presented an extremely rare case of lung abscess following bronchoscopy associated with lung cancer that extended directly into the chest wall. CASE PRESENTATION: A 49-year-old man with adenocarcinoma underwent bronchoscopy. Eight days after the biopsy, the patient presented with chills and anterior chest wall pain. Chest computed tomography (CT) scan revealed a gas-containing lung abscess, measuring 10 cm in the left upper lobe and subcutaneous emphysema. The coronal view of the CT indicated a continuous passage of air from the lung abscess to the subcutaneous emphysema beneath the pectoralis muscle. Surgical debridement of the subcutaneous abscess was performed, resulting in drainage of a large volume of purulent material. We confirmed that the lung abscess had directly extended to the chest wall, leading to a decision to perform segmentectomy of the upper division of the left lung. CLINICAL DISCUSSION: Lung abscess associated with lung cancer is a rare, life-threatening complication, which may lead to significant delays in the commencement of oncological treatment and potentially worsen long-term outcomes. In the present case, surgical findings confirmed a lung abscess extending directly to the chest wall. Surgical therapy is the treatment of choice for this rare condition, providing rapid focus control. Therefore, prompt initiation of surgical therapy is essential when conservative measures prove ineffective. CONCLUSION: Lung abscesses may extend into the chest wall during differential diagnosis of infectious diseases of the chest wall. Successful treatment of this rare condition depends on prompt surgical intervention.

Age distribution and disease severity of COVID-19 patients continued to change in a time-dependent manner from May 2021 to April 2022 in the regional core hospital in Japan.

The present retrospective study aimed to examine the real-world data regarding time-dependent changes in the age distribution of patients with coronavirus disease 2019 (COVID-19) as well as the severity and infectivity in a regional core hospital in Japan. Patients with COVID-19 who visited the fever outpatient branch in Takagi Hospital during phase I (May 1 to December 31, 2021), and during phase II (January 1 to April 30, 2022) were evaluated. The age distribution of outpatients and the characteristics of inpatients aged > 75 years were compared between phases I and II. The age distribution of outpatients shifted from the older generation in phase I to the younger generation in phase II (p < 0.01). Disease severity might be reduced in a time-dependent manner with a decrease in the hospitalization rate (phase I: 145/368 (39.4%); phase II: 104/1496 (7.0%); p < 0.01) and mortality rate (phase I: 10/368 (2.7%); phase II: 7/1496 (0.5%); p < 0.01). The number of patients increased in phase II (374.0/month) compared to that in phase I (36.8/month). Regarding the older inpatients, the disease severity of COVID-19 and hospitalization days were reduced in phase II compared to those in phase I (p < 0.01, each). In conclusion, the present study suggests a change in the age distribution of patients with COVID-19, a decrease in toxicity, and an increase in infectivity of severe acute respiratory syndrome coronavirus 2 in a time-dependent manner.

The evolutionarily conserved G protein-coupled receptor SREB2/GPR85 influences brain size, behavior, and vulnerability to schizophrenia.

The G protein-coupled receptor (GPCR) family is highly diversified and involved in many forms of information processing. SREB2 (GPR85) is the most conserved GPCR throughout vertebrate evolution and is expressed abundantly in brain structures exhibiting high levels of plasticity, e.g., the hippocampal dentate gyrus. Here, we show that SREB2 is involved in determining brain size, modulating diverse behaviors, and potentially in vulnerability to schizophrenia. Mild overexpression of SREB2 caused significant brain weight reduction and ventricular enlargement in transgenic (Tg) mice as well as behavioral abnormalities mirroring psychiatric disorders, e.g., decreased social interaction, abnormal sensorimotor gating, and impaired memory. SREB2 KO mice showed a reciprocal phenotype, a significant increase in brain weight accompanying a trend toward enhanced memory without apparent other behavioral abnormalities. In both Tg and KO mice, no gross malformation of brain structures was observed. Because of phenotypic overlap between SREB2 Tg mice and schizophrenia, we sought a possible link between the two. Minor alleles of two SREB2 SNPs, located in intron 2 and in the 3' UTR, were overtransmitted to schizophrenia patients in a family-based sample and showed an allele load association with reduced hippocampal gray matter volume in patients. Our data implicate SREB2 as a potential risk factor for psychiatric disorders and its pathway as a target for psychiatric therapy.

[Effective glucocorticoid treatment in a case of idiopathic fibrosing mediastinitis].

A 54-year-old man was admitted to Saga University hospital with dyspnea on effort and a sensation of pressure in the chest. Chest CT images showed a low-density mass in the mediastinum surrounding the carina and left hilus, causing narrowing of both the left pulmonary artery and left main bronchus. The pathological findings from a surgical biopsy showed markedly fibrotic tissue with lymphocytes and plasmacytes, and we diagnosed idiopathic fibrosing mediastinitis, stage II. Oral glucocorticoid treatment of 30 mg/day prednisolone reduced the mass and improved the narrowing of the left pulmonary artery and left main bronchus. The patient was given low-dosage glucocorticoids as maintenance treatment. Previous reports indicated that idiopathic fibrosing mediastinitis with severe tissue fibrosis is difficult to control with glucocorticoid monotherapy. Here, we report a case of idiopathic fibrosing mediastinitis that was effectively treated with glucocorticoids.

Clinico-radiologic Characteristics of Pulmonary Visceral Larva Migrans Caused by Ascaris suum.

Objective Visceral larva migrans (VLM) caused by Ascaris suum is a major health problem in pig farming regions. The clinical characteristics of pulmonary VLM caused by A. suum, however, are unclear. We assessed the clinico-radiologic features of this disease. Methods Medical records, including the results of chest radiography and high-resolution computed tomography (HRCT), were retrospectively reviewed from January 2000 through June 2019, at the University of Miyazaki Hospital and Kyoritsuiin Hospital in Miyazaki Prefecture, Japan. Results Seven patients with VLM caused by A. suum were identified. All seven patients had a unique habit of consuming raw foods, such as organic vegetables, chicken, turkey, wild boar, and venison. All but one patient, who had eosinophilic pneumonia with a fever and severe fatigue, had only mild or no respiratory symptoms. All 7 patients had remarkable eosinophilia (median, 1,960/µL) and high serum IgE levels (median, 1,346 IU/mL). Chest HRCT revealed multiple nodules and multiple nodular ground-glass opacities in 57% and 29% of the patients, respectively. The pulmonary lesions were located predominantly in subpleural areas. All seven patients were treated with albendazole, which led to improvement within two to three months. Neither eggs nor parasites were detected in the feces or sputum of any patient. Conclusion Consumption of raw organic vegetables or raw meat is a possible route of A. suum infection. Infected patients exhibit mild respiratory symptoms, and multiple nodules with a halo in the subpleural area are a common finding on chest HRCT. Treatment with albendazole was effective in these cases.

Significance of nuclear LOXL2 inhibition in fibroblasts and myofibroblasts in the fibrotic process of acute respiratory distress syndrome.

Fibrotic scarring is an important prognostic factor of acute respiratory distress syndrome (ARDS). There are currently no antifibrotic drugs or other therapeutic agents for ARDS. Lysyl oxidase-like 2 (LOXL2), an amine oxidase, contributes to fibrotic scarring by facilitating collagen cross-linking. Recent clinical trials revealed that a monoclonal inhibitory antibody against LOXL2 failed to show benefit over placebo in patients with fibrotic disorders involving the lungs. These clinical results raise the possibility that targeting the extracellular enzymic activity of LOXL2 is not in itself sufficient to prevent fibrotic scarring. We investigated the role of LOXL2 in the pathogenesis of ARDS in vivo, in vitro, and in samples from patients with ARDS. After lung injury, LOXL2 was unevenly expressed in the nuclei of lung fibroblasts and myofibroblasts in the fibrotic phase. Nuclear LOXL2 expression was upregulated in lung fibroblasts after transforming growth factor-beta1 (TGF-ß1)-treatment. LOXL2 silencing abrogated the TGF-ß1-induced expression of a myofibrogenic-progenitor marker, the appearance of proto-myofibroblasts, and the evolution of differentiated myofibroblasts in lung fibroblasts. Nuclear upregulation of Snail was evident in myofibroblasts during the fibrotic phase after lung injury. We detected high levels of LOXL2 protein in the lungs of ARDS patients, specifically during the proliferative and fibrotic phases. Our results highlight nuclear LOXL2 in fibroblasts as a primary causative driver of cell-fate decision toward myofibroblasts and of the progression of fibrotic scarring. A nuclear-LOXL2-targeted agent could be a promising therapeutic strategy against fibrotic disorders including ARDS.

[A case of mucormycosis of the thoracic wall in a non-immunocompromised man].

An 80-year-old man visited our hospital for the treatment of an anterior chest wall eruption from February 2007 and presented with dull pain in August 2007. He was referred to our department because chest CT showed the formation of an abscess from the subcutaneous area to the thoracic wall. Histological findings obtained from CT-guided biopsy revealed epithelioid granuloma without caseous necrosis, but both acid-fast bacteria and bacteriologic culture obtained from aspirated fluid samples were negative. Antituberculous therapy was selected because a tuberculous abscess was strongly suspected. However, the patient discontinued treatment soon after therapy began. He visited our hospital again for chest pain due to rupture of the abscess in October 2007. The pathological findings obtained from a second biopsy gave the same results, and antituberculosis therapy was restarted. However, his CT findings had worsened by August 2008, and a third biopsy was performed. Histopathologically, we diagnosed mucormycosis based on the findings of fungal hyphae, with broad, irregular branching at right angles. Thereafter, liposomal amphotericin B (L-AMB) was given intravenously and the abscess markedly improved. Excision was then performed, followed by adjuvant L-AMB administration, and there has been no recurrence to date.

The dopaminergic stabilizer ASP2314/ACR16 selectively interacts with D2(High) receptors.

Dopaminergic stabilizers are recognized as compounds that can either enhance or antagonize dopamine (DA)-dependent behaviors depending on the prevailing dopaminergic tone. The dopaminergic stabilizer ASP2314 is being tested clinically and has been reported to have antipsychotic effects in a clinical trial as an add on medication. To elucidate the mechanisms of action of this dopaminergic stabilizer, its potency on the functional dopamine D2(High) receptors was examined. In competition with D2 receptors selectively labeled by [3H]domperidone, ASP2314 had a dissociation constant, Ki(High), of 1.62 microM for D2(High) in human cloned D2Long receptors and 0.83 muM for rat homogenized striata. Using the D2 agonist ligand [3H](+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol ((+)PHNO), ASP2314 had a high-affinity Ki of 32 nM for D2(High) for rat homogenized striata. ASP2314 stimulated the incorporation of [35S]GTP-gamma-S into rat striata by 50% at 43 nM, and into the cloned D2Long membranes by 50% at 3.2 microM (compared to 100% stimulation by 10 microM dopamine). With similar concentrations of ASP2314 inhibiting the binding of ligands at D2(High) and stimulating [35S]GTP-gamma-S incorporation, the data indicate that the dopaminergic stabilizing action of ASP2314 may be related to the selectivity for the D2(high) state of the D2 receptor.

Allergic bronchopulmonary aspergillosis complicated by eosinophilic chronic rhinosinusitis successfully treated with mepolizumab.

A 67-year-old woman was admitted to our hospital because of frequent asthma attacks and refractory chronic rhinosinusitis. She was diagnosed with allergic bronchopulmonary aspergillosis (ABPA) concomitant with eosinophilic chronic rhinosinusitis (ECRS) on the basis of peripheral blood eosinophilia, precipitating antibodies against Aspergillus fumigatus, elevated total serum IgE, pulmonary infiltration, central bronchiectasis, mucoid impaction, and bilateral rhinosinusitis with nasal polyps, which showed remarkable eosinophil accumulation histologically, especially in the ethmoid sinuses. Treatment with mepolizumab, 100 mg every 4 weeks, was initiated for both the ABPA and ECRS. Consistent with the decrease in the peripheral eosinophil count, the asthma and rhinosinusitis symptoms were drastically ameliorated. Not only her airway symptoms but also her exercise tolerance and pulmonary function test results remarkably improved. Mepolizumab therapy enhanced the quality of life for this patient with intractable ABPA and ECRS.

Desquamative interstitial pneumonia complicated by diffuse alveolar haemorrhage.

We report a rare case of desquamative interstitial pneumonia (DIP) with diffuse alveolar haemorrhage (DAH). A 56-year-old man diagnosed with DIP by surgical lung biopsy 2 years ago was admitted to our hospital because of severe acute respiratory failure. The DIP had progressed despite smoking cessation. On admission, the patient appeared extremely ill, and physical examination revealed respiratory distress. The patient required mechanical ventilation. High-resolution computed tomography showed diffuse ground glass opacity in both lungs. The bronchoalveolar lavage fluid was bloody, and numerous haemosiderin-laden alveolar macrophages were detected. Pulse steroid therapy followed by oral prednisolone immediately relieved the respiratory failure and improved the long-term control of the DIP. Paired sera tests confirmed the diagnosis of influenza A/H3N2 virus infection, which was the cause of the DAH. Chronically progressive DIP with acute respiratory failure due to DAH was successfully treated by steroid therapy.

Human immunodeficiency virus type 1 Vpr induces cell cycle arrest at the G(1) phase and apoptosis via disruption of mitochondrial function in rodent cells.

Vpr of human immunodeficiency virus type 1 causes cell cycle arrest at the G(2)/M phase and induces apoptosis after G(2)/M arrest in primate cells. We have reported previously that Vpr also induces apoptosis independently of G(2)/M arrest in human HeLa cells. By contrast, Vpr does not induce G(2)/M arrest in rodent cells, but it retards cell growth. To clarify the relationship between cell cycle arrest and apoptosis, we expressed Vpr endogenously in rodent cells and investigated cell cycle profiles and apoptosis. We show here that Vpr induces cell cycle arrest at the G(1) phase and apoptosis in rodent cells. Vpr increased the activity of caspase-3 and caspase-9, but not of caspase-8. Moreover, Vpr-induced apoptosis could be inhibited by inhibitors of caspase-3 and caspase-9, but not by inhibitor of caspase-8. We also showed that Vpr induces the release of cytochrome c from mitochondria into the cytosol and disrupts the mitochondrial transmembrane potential. Finally, we showed that apoptosis occurred in HeLa cells through an identical pathway. These results suggest that disruption of mitochondrial functions by Vpr induces apoptosis via cell cycle arrest at G(1), but that apoptosis is independent of G(2)/M arrest. Furthermore, it appears that Vpr acts species-specifically with respect to induction of cell cycle arrest but not of apoptosis.

Unfavorable peripheral intravenous catheter replacements can be reduced using an integrated closed intravenous catheter system.

PURPOSE: To evaluate the clinical usefulness of an integrated closed intravenous catheter system (CICS) with a preattached stabilization platform and extension tube (BD Nexiva™; Becton, Dickinson and Company) in Japanese patients. METHODS: In this open, single-center study, patients who required peripheral intravenous (PIV) catheterization for ≥72 hours were quasi-randomized to receive a CICS or a conventional intravenous catheter. Study outcomes included adverse events during catheter insertion, catheter replacements during the initial 72 hours, catheter survival rate at 72 hours after insertion and costs of initial catheterization and catheter replacement. RESULTS: Of 359 patients enrolled, 194 received the CICS and 165 received the conventional catheter. The incidence rates of ≥1 failed insertion attempts, blood leakage and blood exposure were similar in both groups. The survival rate of the CICS group (83.7%) was significantly higher than that of the conventional catheter group (62.6%) in the intention-to-treat analysis (p=0.0085). There were significantly fewer catheter replacements due to catheter-related complications (e.g., catheter failure or extravasation) in the CICS group (p=0.0056). Although the initial cost per patient was greater for the CICS group (US$17.07 vs. US$13.26), the total cost per patient over 72 hours was similar (US$21.00 vs. US$20.30) because of the cost of unplanned replacements of conventional catheters. CONCLUSIONS: Although rates of adverse events at insertion were similar for both catheters, significantly fewer patients required unplanned reinsertion with the CICS. The results suggest that the longer survival rate for the CICS can offset the higher initial catheterization costs.

Gq/11-induced intracellular calcium mobilization mediates Per2 acute induction in Rat-1 fibroblasts.

Phase resetting is one of the essential properties of circadian clocks that is required for the adjustment to a particular environment and the induction of Per1 and Per2 clock genes is believed to be a primary molecular event during this process. Although the intracellular signal transduction pathway underlying Per1 gene activation has been well characterized, the mechanisms that control Per2 up-regulation have not yet been elucidated. In our present study, we demonstrate that Gq/11 coupled receptors mediate serum-induced immediate rat Per2 (rPer2) transactivation in Rat-1 fibroblasts via intracellular Ca2+ mobilization. Stimulation of these cells with a high concentration of serum was found to rapidly increase the intracellular Ca2+ levels and strongly up-regulated rPer2 gene. rPer2 induction by serum stimulation was abrogated by intracellular Ca2+ chelation and depletion of intracellular Ca2+ store, which suggests that the calcium mobilization is necessary for the up-regulation of rPer2 gene. In addition, suppression of Gq/11 function was observed to inhibit both Ca2+ mobilization and rPer2 induction. Further, we demonstrated that endothelin-induced acute rPer2 transactivation via Gq/11-coupled endothelin receptors is also suppressed by a Gq/11 specific inhibitor. These findings together suggest that serum and endothelin utilize a common Gq/11-PLC mediated pathway for the transactivation of rPer2, which involves the mobilization of calcium from the intracellular calcium store.

Abnormal development of the olfactory bulb and reproductive system in mice lacking prokineticin receptor PKR2.

Prokineticins, multifunctional secreted proteins, activate two endogenous G protein-coupled receptors PKR1 and PKR2. From in situ analysis of the mouse brain, we discovered that PKR2 is predominantly expressed in the olfactory bulb (OB). To examine the role of PKR2 in the OB, we created PKR1- and PKR2-gene-disrupted mice (Pkr1(-/-) and Pkr2(-/-), respectively). Phenotypic analysis indicated that not Pkr1(-/-)but Pkr2(-/-)mice exhibited hypoplasia of the OB. This abnormality was observed in the early developmental stages of fetal OB in the Pkr2(-/-) mice. In addition, the Pkr2(-/-) mice showed severe atrophy of the reproductive system, including the testis, ovary, uterus, vagina, and mammary gland. In the Pkr2(-/-) mice, the plasma levels of testosterone and follicle-stimulating hormone were decreased, and the mRNA transcription levels of gonadotropin-releasing hormone in the hypothalamus and luteinizing hormone and follicle-stimulating hormone in the pituitary were also significantly reduced. Immunohistochemical analysis revealed that gonadotropin-releasing hormone neurons were absent in the hypothalamus in the Pkr2(-/-) mice. The phenotype of the Pkr2(-/-) mice showed similarity to the clinical features of Kallmann syndrome, a human disease characterized by association of hypogonadotropic hypogonadism and anosmia. Our current findings demonstrated that physiological activation of PKR2 is essential for normal development of the OB and sexual maturation.