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Background Aims: The prognostic impact of platelet recovery after autologous hematopoietic cell transplantation (AHCT) on clinical outcomes remains to be elucidated. We aimed to clarify the impact of platelet recovery on clinical outcomes, risk factors of delayed platelet recovery and the necessary dose of CD34 + cells for prompt platelet recovery in each patient., Methods: Using a nationwide Japanese registry database, we retrospectively analyzed clinical outcomes of 5222 patients with aggressive non-Hodgkin lymphoma (NHL) or multiple myeloma (MM)., Results: At a landmark of 28 days after AHCT, a delay of platelet recovery was observed in 1102 patients (21.1%). Prompt platelet recovery was significantly associated with superior overall survival (hazard ratio [HR] 0.32, P < 0.001), progression-free survival (HR 0.48, P < 0.001) and decreased risks of disease progression (HR 0.66, P < 0.001) and non-relapse/non-progression mortality (HR 0.19, P < 0.001). The adverse impacts of a delay of platelet recovery seemed to be more apparent in NHL. In addition to the dose of CD34 + cells/kg, disease status, performance status and the hematopoietic cell transplant-specific comorbidity index in both diseases were associated with platelet recovery. We then stratified the patients into three risk groups according to these factors. For the purpose of achieving 70% platelet recovery by 28 days in NHL, the low-, intermediate- and high-risk groups needed more than 2.0, 3.0 and 4.0 × 10 6 CD34 + cells/kg, respectively. In MM, the low-risk group needed approximately 1.5 × 10 6 CD34 + cells/kg, whereas the intermediate- and high-risk groups required 2.0 and 2.5 × 10 6 CD34 + cells/kg to achieve about 80% platelet recovery by 28 days., Conclusions: A delay of platelet recovery after AHCT was associated with inferior survival outcomes., Competing Interests: Declaration of Competing Interest HN has received honoraria from Otsuka Pharmaceutical, Bristol-Myers Squibb, Pfizer, Novartis, Takeda Pharmaceuticals, Celgene, Janssen Pharmaceuticals, Eisai, Chugai Pharmaceutical Co., Sanofi, Meiji Seika Pharma and Nippon Shinyaku., (Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Breast screening services were suspended for several months owing to the coronavirus disease 2019 (COVID-19) pandemic. We estimated the potential impact on breast cancer mortality using long-term global observations. However, the magnitude of the impact may vary across countries; therefore, we conducted an analysis and modeling study of this impact in Japan., Patients and Methods: We compared the clinicopathological features of breast cancers between the nonpandemicgroup (April 1, 2019 to October 31, 2019) and the pandemic group (April 1, 2020 to October 31, 2020). We also compared the estimated 10-year survival rates between the two groups based on the weighted average of the 10-year survival rate by clinical stage and site (2004-2007)., Results: Results...Pandemic-related disruption decreased the number of breast cancer cases from296 to 249 during both 7-month periods. The percentage of patients with stage IIB or higher disease was significantly higher in the pandemic group than in the non-pandemic group (22.0% vs. 31.3%, P = 0.0133). The percentage of cases with a Ki-67 labeling index higher than 20% tended to be higher in the pandemic group than in the non-pandemic group (62.2% vs. 54.4%). The estimated 10-year survival rate was lower in the pandemic group than in the non-pandemic group (83.9% vs. 87.9%, 95% confidence interval of the difference: 0.87-8.8, P > 0.05)., Conclusion: We found more aggressive and advanced disease afterthe suspension of breast cancer screening services owing to the COVID-19 pandemic. This may have affected the long-term clinical outcomes of patients with breast cancer., (Copyright © 2023. Published by Elsevier Inc.)

Aims: The relationship between stress to endoplasmic reticulum (ER) and periodontitis has been known, and ER stress induced by Porphyromonas gingivalis results in the loss of alveolar bone. Salubrinal is a small synthetic compound and attenuates ER stress through inhibition of de-phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). In this study, we examined whether salubrinal attenuates periodontitis in a mouse model of experimental periodontal disease., Materials and Methods: We evaluated loss of alveolar bone and attachment levels in periodontium using micro-computed tomography (μCT) and hematoxylin-eosin (HE) staining, respectively. Furthermore, we measured osteoclast numbers using tartrate-resistant acid phosphatase (TRAP) staining and osteoblast numbers using HE staining for bone resorption and for bone formation, respectively. To examine the inhibitory effects of salubrinal against pro-inflammatory cytokines, we measured TNF-α and IL1-β score in periodontium using immunohistostaining., Key Findings: The results revealed that salubrinal suppressed loss of alveolar bone and attachment levels in periodontium induced by periodontitis. It decreased osteoclast numbers and increased osteoblasts. It also suppressed the expression levels of TNF-α in periodontium., Significance: These results show that salubrinal alleviates periodontitis through suppression of alveolar bone resorption and the pro-inflammatory cytokine, and promotion of the bone formation. Since salubrinal has been shown to have these beneficial effects for periodontal disease, it may provide a novel therapeutic possibility for the disease., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Monocyte-derived fibrocytes recently garnered attention because the novel pathogenesis of myelofibrosis (MF), and suppression of fibrocyte differentiation by serum amyloid P remarkably improved MF. We previously revealed that human fibrocytes highly expressed signaling lymphocytic activation molecule F7 (SLAMF7) compared with macrophages and that SLAMF7 high monocytes in the peripheral blood (PB) of MF patients were significantly elevated relative to those in healthy controls (HCs). In this study, we evaluated SLAMF7 high monocyte percentage in the PB of HCs, myeloproliferative neoplasm (MPN) patients with MF, and MPN patients without MF by using a cross-sectional approach. We found that MPN patients with MF who harbored JAK2 V617F had a significantly elevated SLAMF7 high monocyte percentage, which correlated positively with the JAK2 V617F allele burden. In addition, the serum concentration of interleukin-1ra (IL-1ra) was significantly correlated with the SLAMF7 high monocyte percentage and JAK2 V617F allele burden. These findings suggest that both SLAMF7 high monocytes and IL-1ra could be useful noninvasive markers of MF onset. Furthermore, the JAK2 V617F allele burden of SLAMF7 high monocytes was significantly higher than that of SLAMF7 low monocytes and could be a potential target of elotuzumab (Elo), an anti-SLAMF7 antibody used for treating multiple myeloma. Elo independently inhibited differentiation of fibrocytes derived not only from HCs but also from MF patients in vitro. Elo also ameliorated MF and splenomegaly induced by romiplostim administration in humanized NOG mice. In conclusion, an increase of SLAMF7 high monocytes with higher JAK2 V617F allele burden was associated with the onset of MF in MPN patients harboring JAK2 V617F, and Elo could be a therapeutic agent for MPN patients with MF who harbor JAK2 V617F., (© 2019 by The American Society of Hematology.)

Background: The cerebroprotective effect of retrograde cerebral perfusion (RCP) and selective antegrade cerebral perfusion (SCP) still remains controversial. Laser speckle flowgraphy has shown much promise for novel perioperative neuromonitoring by assessing blood flow of the optic nerve head. This study aimed to evaluate the cerebral microcirculation in humans using laser speckle flowgraphy during simple circulatory arrest, RCP, and SCP under moderate hypothermia and to investigate whether RCP under moderate hypothermia is a reliable method of cerebral protection., Methods: A total of 23 consecutive patients who underwent a scheduled aortic arch or hemiarch surgical procedure on thoracic aorta aneurysm were enrolled. The laser speckle flowgraphy measurement that calculates mean blur ratio, a parameter of cerebral circulation, was obtained 6 times: after induction of anesthesia, baseline (T1), after initiation of cardiopulmonary bypass with cardiac arrest (T2), simple circulatory arrest (T3), RCP (T4), SCP (T5), and after the termination of cardiopulmonary bypass (T6)., Results: Both mean blur ratios of simple circulatory arrest and RCP were significantly decreased compared with baseline. In contrast, no significant differences were observed between simple circulatory arrest and RCP. The mean blur ratio of SCP was significantly increased compared with both simple circulatory arrest and RCP., Conclusions: In conclusion, no significant difference was observed in the cerebral circulation between RCP and simple circulatory arrest without adjunctive strategy under moderate hypothermia. In contrast, the cerebral circulation during SCP was significantly higher than simple circulatory arrest and RCP. These results suggest that cerebral microcirculation may not be adequate during RCP compared with SCP under moderate hypothermia., (Copyright © 2019 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

The aim of the present study was to characterize the effect of long-term usage of dienogest, a fourth-generation progestin that possesses progestogen and anti-androgen activities, on the stockpile of oocytes and fertility after administration. Female ICR mice (100 days old) were divided into a dienogest group and a control group. The mice received 16 consecutive subcutaneous injections of 5 mg dienogest dissolved in corn oil or corn oil as a vehicle control every 4 days. The mice treated with dienogest had more total offspring and larger litter sizes after the final administration than the mice treated with the vehicle control. Greater numbers of primordial follicles were detected at both 4 and 80 days after the final administration. No significant differences were found in serum anti-Müllerian hormone concentrations at 4 and 80 days after the final dienogest administration. The ratio of primary to primordial follicles was decreased in 3-day-old newborn ovaries cultured for 4 days with dienogest (10 -7 , 10 -6 and 10 -5 mol/l) compared with ovaries cultured without dienogest. The results of the present study indicate that dienogest suppresses the activation of primordial follicles during its administration and preserves the primordial follicle stockpile and subsequent fertility in mice., (Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

When the intercostal and lumbar arteries are occluded by plaque or thrombus, spinal cord perfusion depends on collateral circulation. Some reports have demonstrated collateral circulation to the artery of Adamkiewicz via computed tomography and magnetic resonance angiographies. However, intraspinal collateral circulation to the artery of Adamkiewicz along the spinal cord has not been reported previously. Here, we report two patients with intraspinal collateral circulation to the artery of Adamkiewicz along the spinal cord that was detected with intra-arterial injected computed tomography angiography., (Copyright © 2016 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Introduction: The purpose of this study was to analyze the high-resolution computed tomography (HRCT) features of lung carcinoma on the basis of epidermal growth factor receptor gene (EGFR) mutation status., Methods: A total of 263 consecutive patients in whom lung adenocarcinoma was diagnosed at our institution between January 2010 and December 2011 were enrolled in the study. All patients underwent HRCT and analysis of EGFR mutation status. The HRCT findings were retrospectively analyzed for tumor size, multiple bilateral lung metastases, convergence of surrounding structures, surrounding ground-glass opacity, prominent peribronchovascular extension, air bronchogram, notch, pleural indentation, spiculation, cavity, and pleural effusions., Results: EGFR mutations were demonstrated in 103 patients (39.2%); the remaining 160 patients (60.8%) had the nonmutated type of adenocarcinoma. Compared with the nonmutated group, the mutated group had significantly higher frequencies of multiple bilateral lung metastases (p = 0.0152), convergence of surrounding structures (p < 0.0001), ground-glass opacity (p = 0.0011), and notch (p = 0.0428), but significantly lower frequencies of cavitation (p = 0.0004) and pleural effusions (p = 0.0064). The frequencies of the other CT findings were similar between the two groups. The devised prediction HRCT score for EGFR mutation was 78.4% sensitive and 70.4% specific., Conclusions: EGFR-mutated adenocarcinoma showed significantly higher frequencies of multiple bilateral lung metastases, convergence of surrounding structures, surrounding ground glass opacity, and notch at HRCT compared with the non-EGFR-mutated type. Conversely, EGFR-mutated adenocarcinoma showed cavity and pleural effusions less frequently than the nonmutated type did., (Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Maternal overnutrition (e.g., high-fat (HF) diet) during pregnancy and lactation is believed to cause oxidative stress and increase the risk of metabolic syndrome in offspring. In the present study, we investigated the effects of both maternal and offspring HF diets on metabolic syndrome risk and oxidative stress profiles in mice. Dams of the C57BL/6J mouse strain were fed a HF or control (CO) diet during gestation and lactation. Offspring were weaned at 3weeks of age. The female offspring were sacrificed at weaning, while the males were maintained on the HF or CO diet until 11weeks of age. Tissue samples, including those from liver, were collected from offspring at 3 and 11weeks of age, and lipids, phosphatidylcholine hydroperoxide (PCOOH, an oxidative stress marker), and gene expression were evaluated. Accumulation of lipids, but not PCOOH, was found in the livers of 3-week-old offspring from dams fed the HF diet. When the offspring were maintained on a HF diet until 11weeks of age, marked accumulation of both liver lipids and PCOOH was observed. PCOOH manifestation was supported by the expression of genes such as Gpx4, encoding a PCOOH degrading enzyme. These results suggest that the combination of maternal and offspring overnutrition causes marked oxidative stress in offspring, which accelerates metabolic syndrome. The present findings in offspring from infancy to adulthood may be useful for better understanding the cause-and-effect relationships between oxidative stress and metabolic syndrome development., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Adaptive statistical iterative reconstruction (ASIR) has been used to reduce radiation dose in cardiac computed tomography. However, change of image parameters by ASIR as compared to filtered back projection (FBP) may influence quantification of coronary calcium., Objective: To investigate the influence of ASIR on calcium quantification in comparison to FBP., Methods: In 352 patients, CT images were reconstructed using FBP alone, FBP combined with ASIR 30%, 50%, 70%, and ASIR 100% based on the same raw data. Image noise, plaque density, Agatston scores and calcium volumes were compared among the techniques., Results: Image noise, Agatston score, and calcium volume decreased significantly with ASIR compared to FBP (each P < 0.001). Use of ASIR reduced Agatston score by 10.5% to 31.0%. In calcified plaques both of patients and a phantom, ASIR decreased maximum CT values and calcified plaque size., Conclusion: In comparison to FBP, adaptive statistical iterative reconstruction (ASIR) may significantly decrease Agatston scores and calcium volumes., (Copyright © 2016 Society of Cardiovascular Computed Tomography. Published by Elsevier Inc. All rights reserved.)

The single-crystal rotation technique was applied to magnetically oriented microcrystal arrays (MOMAs) of cellobiose (monoclinic) to determine the principal values and principal axes of the chemical shift tensors of C1 and C1' carbons. Rotations were performed about the magnetic χ1, χ2, and χ3 axes of MOMA, and the measurements were taken at six different orientations with respect to the applied magnetic field. Under these rotations, crowded peaks were reduced and the peaks for the C1 and C1' carbons were identified by comparing with simulation results. Six components of the chemical shift tensor expressed with respect to the magnetic χ1χ2χ3-frame were determined. The tensors thus obtained were transformed into those relative to the molecular frame., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: To evaluate the factors related to the choice of a tracheostomy and invasive ventilation in amyotrophic lateral sclerosis patients and to determine survival time after a tracheostomy at a single institute in Japan between 1990 and 2010., Methods: Data for survival time until death or tracheostomy were obtained from 160 patients. Fifty-two patients (33%) underwent tracheostomy/mechanical ventilation., Results: Tracheostomy and invasive ventilation prolonged median survival time (74 months), as did non-invasive ventilation (48 months) when compared to a non-ventilation-supported control group (32 months; p<0.001 each). The ratio of tracheostomy/mechanical ventilation in patients >65 years old significantly increased after 1999 (27%) compared to earlier years (10%, p=0.002). Cox proportional modeling confirmed an age of ≤65 years as advantageous for long-term survival after a tracheostomy. In univariate logistic regression analysis, factors related to the decision to perform a tracheostomy included an age of ≤65 years, greater use of non-invasive ventilation, the presence of a spouse, interval and speed from disease onset to diagnosis/tracheostomy and preservation of motor function. In multivariate logistic regression analysis, age, shorter duration from disease onset until tracheostomy and the presence of a spouse were independently associated with the decision to perform a tracheostomy. Kaplan-Meier plots revealed longer survival times in patients who resided at home after a tracheostomy compared to patients who stayed at a hospital (p=0.007)., Conclusions: Tracheostomy and invasive ventilation are frequently used in Japan. Various factors impact patients' decisions to have these procedures. This study identified factors related to the decision-making process and post-tracheostomy survival., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We retrospectively evaluated pelvic magnetic resonance imaging including diffusion-weighted imaging (DWI) of 16 ovarian lesions (5 adenocarcinofibromas, 2 borderline adenofibromas, and 9 benign adenofibromas). All adenocarcinofibromas were detected as large solid areas of strong high signal on DWI, and seven of nine benign adenofibromas and both borderline adenofibromas demonstrated no areas of high signal or small areas of weak high signal. Solid components that appear as areas of strong high signal on DWI might represent a characteristic finding of adenocarcinofibromas., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A woman aged 64 years with cardiac sarcoidosis responded favourably to corticosteroid therapy in terms of recovered longitudinal myocardial strain, as evaluated by strain-encoded magnetic resonance imaging (SENC-MRI). In contrast, circumferential myocardial strain and late gadolinium enhancement demonstrated minimal improvement, suggesting relatively advanced pathology of the myocardial middle layer. We propose SENC-MRI as a marker of disease at an early stage of cardiac sarcoidosis., (Copyright © 2013 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.)

Background: The accuracy of electrocardiogram-gated CT for diagnosing arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is uncertain., Objective: We propose a comprehensive system for scoring characteristic CT findings to diagnose ARVC/D and discuss its utility., Methods: Seventy-seven patients (mean age, 43.1 years; 48 male) diagnosed with ARVC/D or with suspected ARVC/D on the basis of ventricular tachyarrhythmias underwent CT with and without contrast enhancement. We retrospectively graded characteristic CT findings (fatty tissue, bulging appearance, and dilatation of the right ventricle) as minor (1 point) or major (2 points) and then validated our scoring system's utility for diagnosing ARVC/D with the use of the modified 2010 Task Force criteria as the reference standard., Results: We diagnosed 27 of 77 patients with ARVC/D (23 definite, 4 borderline), observing each CT finding more often in patients with ARVC/D than in patients without ARVC/D: fatty tissue, 74.1% versus 12.0%; bulging appearance, 74.1% versus 34.0%; and RV dilatation, 92.6% versus 44.0%. However, observation of each finding in both groups could cause false positive diagnosis. Recursive partitioning analysis showed the superiority of our CT scoring system over methods that used each CT finding and CT findings without fatty tissue for distinguishing ARVC/D and non-ARVC/D. For overall (definite and borderline) and definite ARVC/D diagnosis, sensitivities were 77.8% and 87.0%, specificities were 96.0% and 94.4%, positive predictive values were 91.3% and 87.0%, negative predictive values were 88.9% and 94.4%, and accuracies were 89.6% and 92.2%, respectively., Conclusions: Our CT scoring system showed excellent diagnostic ability and might aid differentiation of ARVC/D from ventricular tachyarrhythmias., (Copyright © 2013 Society of Cardiovascular Computed Tomography. Published by Elsevier Inc. All rights reserved.)

A 76-year-old woman came to us because of staggering, fever, dysarthria, and appetite loss. Magnetic resonance imaging (MRI) of the brain revealed multiple masses with surrounding edema. Chest X-ray and computed tomography demonstrated a mass-like lesion in the left lung and left pleural effusion. Lung cancer and multiple brain metastases were suspected. However, the brain lesions demonstrated a high intensity through diffusion-weighted MRI. The finding was an important key to differentiate brain abscesses from lung cancer metastases., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Chemical shift tensors for the carboxyl and methyl carbons of L-alanine crystals were determined using a magnetically oriented microcrystal array (MOMA) prepared from a microcrystalline powder sample of L-alanine. A MOMA is a single-crystal-like composite in which microcrystals are aligned three-dimensionally in a matrix resin. The single-crystal rotation method was applied to the MOMA to determine the principal values and axes of the chemical shift tensors. The result showed good agreement with the literature data for the single crystal of L-alanine. This demonstrates that the present technique is a powerful tool for determining the chemical shift tensor of a crystal from a microcrystal powder sample., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The ability to detect and discriminate sensory stimuli greatly improves with age. To better understand the neural basis of perceptual development, we studied the postnatal development of sensory responses in cortical neurons. Specifically, we analyzed neuronal responses to single-whisker deflections in the posteromedial barrel subfield (PMBSF) of the rat primary somatosensory cortex. Responses of PMBSF neurons showed a long onset latency and duration in the first postnatal week, but became fast and transient over the next few weeks. Trial-by-trial variations of single neuron responses did not change systematically with age, whereas the covariation of responses across trials between neurons (noise correlation) was high on postnatal day 5-6 (P5-6), and gradually decreased with age to near zero by P30-31. Computational analyses showed that pooled responses of multiple neurons became more reliable across stimulus trials with age. The period over which these changes occurred corresponds to the period when rats develop a full set of exploratory whisking behavior. We suggest that reduced noise correlation across a population of neurons, in addition to sharpening the temporal characteristics of single neuron responses, may help improve behavioral performance., (Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Glutamine is the most versatile amino acid and its plasma concentration is the highest of all amino acid. Many transporters are therefore involved in glutamine uptake or efflux. Glutamine is actively released from the placenta into fetal circulation. In this study, we examined the alteration of transporters that transport glutamine into fetal circulation as gestation progresses. High expression levels of system A and y(+)L were found in the rat placenta in the late period of pregnancy and the expression levels of these transporters increased as gestation progressed (p<0.05). On the other hand, the expression of SNAT3, the system N transporter, was detected in the early period of pregnancy and its expression level decreased as gestation progressed (p<0.05). SNAT3 was also found to be expressed in isolated human primary cytotrophoblast cells and its expression level was decreased by their differentiation into syncytiotrophoblast cells (p<0.05). Since this regulation is closely related to glutamine synthetase expression, SNAT3 may play a key role in providing glutamine corresponding to glutamine synthetase function in the early period of gestation. This is the first report on the expression of SNAT3 in the placenta in the early stage of pregnancy.

Palliative care for cancer patients receiving chemotherapy in the outpatient setting is important. The aims of this study were 1) to identify symptom prevalence and intensity in cancer patients receiving chemotherapy and 2) to describe longitudinal follow-up data obtained from repeated assessment using the distress thermometer (DT). Questionnaires were distributed to consecutive cancer outpatients newly starting chemotherapy at the first appointment and at every hospital visit. The questionnaire included the severity of 11 symptoms (M. D. Anderson Symptom Inventory [MDASI], Japanese version), the DT, and the need for help in four psychosocial areas (decision-making, economic problems, nutrition, and daily activities). In total, 4000 questionnaires were returned by 462 patients. The frequently identified problems were oral problems (21%), insomnia (19%), psychological distress (defined as a DT score of 6 or more; 15%), help with information and decision-making (14%), severe fatigue (8.2%), and severe appetite loss (6.3%). Cluster analysis identified four symptom clusters: 1) fatigue and somnolence; 2) pain, dyspnea, and numbness; 3) nausea, appetite loss, and constipation; and 4) psychological distress. Of 165 patients with a DT of score 6 or more, 115 patients (70%) demonstrated a DT score below 6 at a median of 17 days follow-up. In the remaining 50 patients who had a DT score of 6 or more at follow-up, 34 patients (68%) had one or more physical symptoms rated at 7 or more on an 11-point numeric rating scale. Compared with patients with a DT score below 6 at follow-up, patients with a DT score of 6 or more at follow-up had higher levels of all physical symptoms. Frequent symptoms experienced by cancer outpatients receiving chemotherapy may be categorized as: 1) psychosocial issues (insomnia, psychological distress, decision-making support); 2) nutrition-gastrointestinal issues (oral problems, appetite loss, nausea); 3) fatigue; and 4) pain, dyspnea, and numbness. Developing a systematic intervention program targeting these four areas is urgently required. The DT score may be highly influenced by coexisting physical symptoms, and future studies to develop an appropriate system to identify patients with psychiatric comorbidity are necessary.

Peroxidized phospholipid-mediated cytotoxicity is involved in the pathophysiology of many diseases; for example, there is an abnormal increase of phospholipid hydroperoxides in red blood cells (RBCs) of dementia patients. Dietary carotenoids have gained attention as potent inhibitors of RBC phospholipid hydroperoxidation, thereby making them plausible candidates for preventing disease. However, the occurrence of carotenoids in human RBCs is still unclear. This is in contradistinction to plasma carotenoids, which have been investigated thoroughly for analytical methods as well as biological significance. In this study, we developed a method to analyze RBC carotenoids using high-performance liquid chromatography (HPLC) coupled with ultraviolet (UV) diode array detection (DAD) and atmospheric pressure chemical ionization (APCI) mass spectrometry (MS). Under optimized conditions that included extraction, separation, and detection procedures, six carotenoids (lutein, zeaxanthin, beta-cryptoxanthin, alpha-carotene, beta-carotene, and lycopene) were separated, detected by DAD, and concurrently identified based on APCI/MS and UV spectra profiles when an extract from human RBCs was subjected to HPLC-DAD-APCI/MS. The amounts of carotenoids varied markedly (1.3-70.2 nmol/L packed cells), and polar oxygenated carotenoids (xanthophylls) were predominant in RBCs. The HPLC-DAD-APCI/MS method would be a useful tool for clinical studies for evaluating the bioavailability of RBC carotenoids.

Influenza virus infection is an important cause of mortality after hematopoietic stem cell transplantation (HSCT). Although early diagnosis followed by standard therapy with oseltamivir is thought to prevent influenza complications in myeloablative bone marrow transplant recipients, the optimal duration of therapy in these patients has not yet been determined. We describe a case of influenza virus reactivation, after remission had been achieved with standard oseltamivir therapy, in a patient undergoing nonmyeloablative bone marrow transplantation (NMBMT). A rapid diagnostic test (RDT) for influenza virus detection was performed with the ESPLINE Influenza A&B-N kit, which is a rapid, readily available, and widely used approach enabling highly specific and sensitive detection, as well as monitoring, of influenza A and B viruses. However, our case shows that a very low viral load that is undetectable by this RDT can reactivate influenza during the early phase of NMBMT. Our case suggests that oseltamivir administration for influenza infection should be continued, at least until successful engraftment, to prevent virus reactivation. The patient must be frequently and carefully monitored even after the resolution of symptoms and the clearance of viruses from respiratory secretions. The decision to cease oseltamivir therapy safely can be assisted by negative results on reverse transcription polymerase chain reaction (RTPCR) analysis.

To investigate the role of the carbohydrate chain of hen egg ovalbumin (OVA), potential N-glycosylation site-deletion OVA mutants were expressed in yeast. The secretion level of the N292Q and N292/311Q mutants was greatly reduced compared with the wild-type OVA. Furthermore, secretion of the mutants without a carbohydrate chain on Asn-292 could hardly be detected in the culture medium, even if an additional N-glycosylation site was introduced to the OVA molecule. The reduction in secretion level seems to be due to incorrectly folded protein. Moreover, the secretion levels of the wild-type and N311Q mutant reduced in a similar extent as those of the mutants without a carbohydrate chain on Asn-292 in calnexin-disrupted yeast. These results indicate that the carbohydrate chain attached to Asn-292 of OVA has an important role for the secretion and folding in the cells.

Background: It is now well established that vascular inflammation and endothelial dysfunction associated with cardiovascular diseases contributes to insulin resistance., Methods: We investigated the relationship between the homeostasis model assessment-insulin resistance index (HOMA-R) and various serum inflammatory markers and the effect of losartan on serum concentrations of these markers in patients with type 2 diabetes and hypertension. The patients were divided into 2 groups according to the value of HOMA-R with 60 patients with values=2.4 in Group A and 44 patients with values>2.5 in Group B. The variables were measured at baseline and after 6 months of treatment with losartan (50 mg/day)., Results: The HOMA-R concentrations were positively related to TNF-alpha (r=0.336, P<0.01) and inversely related to adiponectin (r=-0.405, P<0.01) and extracellular-superoxide dismutase (EC-SOD) (r=-0.452, P<0.01). Stepwise multiple regression analysis showed a significant relationship between HOMA-R and adiponectin (F=8.74) and EC-SOD (F=14.39). In Group B, losartan treatment significantly increased the serum concentrations of EC-SOD and adiponectin and decreased TNF-alpha and HOMA-R., Conclusion: Serum EC-SOD concentrations may be a sensitive biochemical marker of insulin resistance in patients with type 2 diabetes and hypertension and that losartan improves insulin sensitivity by increasing EC-SOD and adiponectin production and decreasing TNF-alpha production.

We report a case of Stanford type A acute aortic dissection caused by blunt trauma in a patient with situs inversus. A 57-year-old man was involved in an explosion accident. It was indicated that he had suffered Stanford type A acute aortic dissection, cardiac tamponade, and situs inversus by contrast enhanced computed tomography and echocardiography. With the introduction of anesthesia, he went into shock. An extracorporeal circuit was immediately introduced with heart massage. The ascending aorta was replaced with a prosthesis using cardiopulmonary bypass. The branches of the aortic arch were mirror-image reversed. The patient's general condition improved, and he was discharged from our department 3 weeks after the operation.

Background: Sixteen-channel multidetector-row computed tomography (MDCT), with higher spatial and temporal resolution, enables noninvasive visualization of images with reduced motion artifact and breath-holding time. We compared images of 16-channel MDCT and selective bypass graft angiography among patients who had off-pump coronary artery bypass graft surgery., Methods: The study, conducted from April 2003 to March 2004, involved 42 patients who underwent off-pump coronary artery bypass graft surgery. Samples yielded a total of 96 arterial grafts, 5 vein grafts. Sixteen-channel MDCT (LightSpeed Ultra 16; GE Healthcare, Milwaukee, Wisconsin) was performed on each patient using 500-ms or 600-ms rotation time, 0.625-mm slice thickness, and mean scanning time of approximately 24 seconds., Results: If several sequential anastomoses in one graft existed, each was calculated as a separate graft. Selective bypass graft angiography yielded a patency rate of 97% (155 of 160). Multidetector-row computed tomography enabled detection of 143 of 155 patent grafts and all 5 occluded grafts visualized by selective bypass graft angiography (100% sensitivity and 93% specificity for graft occlusion after exclusion of grafts not evaluated by MDCT). In 149 graft anastomoses of 143 patent grafts viewed by MDCT, 2 significant stenoses were detected by both selective bypass graft angiography and MDCT. Twelve distal anastomoses were not evaluated by MDCT because of metallic clip artifacts. Evaluation possible graft anastomoses were 92%. Sensitivity and specificity for significant stenosis after exclusion of graft anastomoses not evaluated by MDCT were 100% and 99%, respectively., Conclusions: High-quality 16-channel MDCT images allowed detection of graft occlusion and significant stenosis of graft anastomosis after off-pump coronary artery bypass graft surgery, demonstrating an alternative tool less invasive than selective bypass graft angiography to assess grafts after surgery.

Background: The early results of emergent coronary artery bypass grafting by conventional operative method for acute myocardial infarction are reported to be poor. The purpose of this study is to evaluate on-pump beating-heart coronary artery bypass grafting for acute myocardial infarction., Methods: Thirty-one patients with acute myocardial infarction underwent emergent surgery between January 1998 and June 2004 at Nayoro City General Hospital. In 16 patients, on-pump surgery was performed on the arrested heart, and in the other 15, on-pump surgery was performed on the beating heart. Early results were compared between the two groups., Results: Preoperative and perioperative patient characteristics revealed no significant differences between the two groups. Although there was no statistically difference between the two groups, the early mortality rates of on-pump arrested-heart coronary bypass grafting (31.3%) was higher than that of on-pump beating-heart coronary bypass grafting (13.3%). Postoperatively, the creatine kinase myocardial band value for the on-pump beating-heart group was significantly lower than that for the on-pump arrested-heart group (221 +/- 200 IU/L versus 666 +/- 540 IU/L, p = 0.008). The incidence of postoperative acute renal failure was significantly higher in the on-pump arrested-heart group than in the on-pump beating-heart group (p = 0.034). The durations of ventilator use and inotropic agent use were longer in the on-pump arrested-heart group than in the on-pump beating-heart group, though the differences were not statistically different (p = 0.152, p = 0.223)., Conclusions: On-pump beating-heart coronary artery bypass grafting has the possibility to eliminate intraoperative global myocardial ischemia and to be an acceptable surgical option for acute myocardial infarction associated with lower postoperative mortality and morbidity.

We determined whether feeding with powdered diet improved the visuospatial ability in female rats by checking the expression of N-methyl-D-aspartate receptor (NMDAR) subunit 1 (NR1) mRNA in the hippocampus. In rats fed standard pelleted diet, males performed better than females in a radial 8-arm maze task as we reported previously. We found that the expression of NR1 mRNA, which may be the key mediator in visuospatial ability in the hippocampus, was also higher in males than in females. However, in rats fed powdered diet, no sex difference was seen in the radial 8-arm maze task and the expression of NR1 mRNA in the hippocampus, since feeding with powdered diet improved the visuospatial ability with increases in the expression of NR1 mRNA in the hippocampus in females. We suggest that the sex difference in visuospatial ability is at least in part due to feeding conditions.

Background/aims: Liver failure associated with infections after hepatectomy remains a cause of mortality. It has recently been reported that toll-like receptor 4 (TLR4) is involved in recognizing lipopolysaccharides (LPS). The aim of this study was to investigate the role of TLR4 in endotoxin-induced liver injury after hepatectomy., Methods: C3H/HeN and C3H/HeJ mice underwent 70% hepatectomy or sham surgery, and LPS was administered 48 h after surgery. Expression of TLR4 mRNA, nuclear factor-kappaB (NF-kappaB) activation, tumor necrosis factor-alpha (TNF-alpha) and serum ALT levels, histological findings, and myeloperoxidase content were examined. Survival after LPS administration was also determined., Results: Hepatic expression of TLR4 was significantly increased 6-72 h after hepatectomy. In mice with endotoxemia after hepatectomy, hepatic NF-kappaB activation was greatly increased. Hepatic mRNA and serum levels of TNF-alpha, and ALT levels were significantly elevated compared with sham operated controls. Focal necrosis with neutrophil infiltration was apparent, which is consistent with increased myeloperoxidase contents in endotoxemia after hepatectomy in C3H/HeN mice. These were completely absent in C3H/HeJ mice. Survival of C3H/HeN mice with endotoxemia after hepatectomy was significantly lower than that of C3H/HeJ mice., Conclusions: Upregulated TLR4 expression and function after hepatectomy plays a pivotal role in endotoxin-induced liver injury after hepatectomy.

We examined the effects of intracerebroventricular injection of neuromedin U (NMU), at a dose that is reported to induce satiety in rats, on the pulsatile luteinizing hormone (LH) secretion in adult ovariectomized (OVX) rats under a normal feeding or a 48-h fasted condition. In OVX rats under the normal feeding condition, injection of NMU (1 nmol/3 microl) significantly decreased the mean LH concentration without affecting the frequency or amplitude of LH pulses, but under the 48-h fasted condition, it significantly decreased the mean LH concentration and the frequency of LH pulses without affecting the amplitude. The interpulse interval was significantly lengthened by NMU injection under the normal and the 48-h fasted condition, but the effect under the 48-h fasted condition was greater than under the normal feeding condition. We also confirmed that the 48-h fasted condition per se did not affect the pulsatile LH secretion in OVX rats. We suggest that NMU and fasting synergistically inhibit the pulsatile LH secretion, even though NMU has been said to act as a satiety factor.

GABA is a potent regulator of gonadotropin release both in male and female rats. We reported 24 h profiles of GABA release in the medial preoptic area (MPO) where gonadotropin-releasing hormone (GnRH) surge generator resides in female rats. In this article, we review the sex difference in 24 h profiles of GABA release. GABA release is high and episodic in male rats without any time dependency, but female rats showed a surge-like secretion of GABA in the early morning of the proestrous day. GABA release rapidly decreased until the afternoon of the day of proestrus followed by the preovulatory luteinizing hormone (LH) surge. The peak time of GABA episodes changes with estrous cycle in female rats. Fitting with the double cosinor method demonstrated that the acrophase of the GABA release in proestrous female rats occurs in the early morning, whereas the acrophases in diestrous females, estrous females and males occur at various time of day. Proestrous female rats showed significant difference in the peak time and acrophase of the GABA release compared with other estrous stages of female and male rats. These results demonstrated further sexual dimorphism of GABA release in the MPO, suggesting that coupling between the GABA release and the circadian clock may be a determining factor in the sex difference of the hypothalamo-pituitary-gonadal (HPG) axis in rats.

We examined the effect of estrogen on the expression of aryl-hydrocarbon receptor (AhR) and two types of AhR nuclear translocator (Arnt1 and Arnt2) mRNAs in the hypothalamus of ovariectomized rats. Northern blotting demonstrated that, in the mediobasal hypothalamus, a subcutaneous injection of 20 microg estradiol benzoate (E(2)) significantly increased the expression of Arnt2 mRNA, but induced no significant changes in the expression of AhR and Arnt1 mRNAs. The expression of Arnt2 mRNA was significantly increased at 4, 24, and 72h after the injection. Immunocytochemical study revealed that the number of Arnt2 immunoreactive cells was also significantly increased at 72h after the injection. Conversely, in the preoptic area, injection of E(2) did not cause significant changes in the expression of any of the three mRNAs. These observations suggest that estrogen regulates Arnt2 expression in the mediobasal hypothalamus and modulates the toxic action of dioxins in rats.

A time production test was performed in seven normal females and seven females with premenstrual syndrome (PMS). We used a time production test for six time intervals (6, 12, 24, 36, 48, 60 s). Time production tests were performed at 08:00, 14:00 and 20:00 h in the follicular, early luteal and late luteal phases (LLP), respectively. The results are as follows. (1) Normal females showed diurnal variations in time production only in the follicular phase (FP). In this phase, they overproduced time at 08:00 h suggesting that the interval clock in normal females was slowest in the morning. (2) Normal females showed menstrual variations in time production only at 20:00 h. They overproduced time in the LLP suggesting that the interval clock in normal females was slowest in the LLP. (3) Neither diurnal nor menstrual variation was found in PMS subjects at any menstrual stage or at any time of the day. These results suggest that the interval timing system in normal females is under the control of ovarian steroid hormones and the circadian clock, but the control of ovarian steroid hormone or the circadian clock does not operate in PMS subjects.

Ghrelin, an endogenous growth hormone (GH) secretagogue, is shown to increase food intake, which action is similar to that of orexin, also a hypothalamic peptide. Since orexin suppresses pulsatile LH secretion in ovariectomized (OVX) rats, the present study was undertaken to investigate whether ghrelin also suppresses LH secretion. Effects of intracerebroventricularly injected ghrelin (0.1 nmol/0.3 microl) were examined in OVX rats treated with a small dose of 17beta-estradiol (E(2)). After ghrelin injection, pulsatile LH secretions which were ongoing in these E(2)-treated OVX rats were significantly suppressed for about 1 h, whereas GH secretion increased, peaking at 30 min. The main parameter suppressed by ghrelin was the pulse frequency, not the pulse amplitude, suggesting the hypothalamus as the site of ghrelin action. This study provides evidence that ghrelin acts not only in the control of food intake but also in the control of LH secretion., (Copyright 2001 Academic Press.)

To determine whether estrogen has an effect on clock genes in the suprachiasmatic nucleus (SCN), we examined the effect of estrogen on the expression of Cry1 and Cry2 mRNAs in the SCN of female rats. Ovariectomized rats were injected with 20 microg 17beta-estradiol at zeitgeber time (ZT) 6 and ZT 18 and killed 24 h after the treatment. Northern blot revealed that the expression of Cry2 mRNA was significantly decreased in the SCN of estrogen-treated rats at both time points [correction]. But estrogen did not affect Cry1 mRNA levels in the SCN at any ZT. These results suggested that Cry1 and Cry2 mRNAs in the SCN were differently regulated by estrogen.

Expressions of Cry1 and Cry2 mRNA in the rat pineal gland were examined by Northern blot. The levels of Cry1 and Cry2 transcript had a marked circadian rhythm with peaks at circadian time (CT) 20 in constant darkness. But the amplitude of the Cry1 rhythm was higher than that of the Cry2 rhythm. Furthermore, a significant increase in Cry1 mRNA levels was caused by light pulse given at CT 16 but not at CT 4, but the expression of Cry2 was not significantly induced by light pulses given at either CT 4 or CT 16. These results suggest that Cry1 in the pineal gland is regulated by photic and circadian information but Cry2 is only regulated by circadian information.

Gonadotropin-releasing hormone (GnRH) neurons are mainly located in the anterior preoptic area (aPOA) and gamma-aminobutyric acid (GABA) is known as a potent regulator of the GnRH neurons. To examine the development of the GABAergic system in the aPOA, immunocytochemistry of glutamic acid decarboxylase 67 (GAD(67)) was performed in immature (postnatal d16, d25 and d30) and mature (postnatal 10 weeks) male rats. All immunocytochemical procedures were simultaneously performed. In the lateral part of the aPOA, the detectable number of GAD(67)-immunoreactive cells was small in the d16 group, but significantly increased in the d25, d30 and mature groups, up to 2.7, 4.8 and 5.7 times the number in the d16 group, respectively. In the diagonal band of Broca (DBB), the number was also small in the d16 group, and significantly increased in the d25, d30 and mature groups upto 1.8, 2.2 and 2.8 times the number in the d16 group, respectively. However, in the cingulate cortex, no significant developmental change was observed. These results suggest that the development of the GABAergic system in the lateral aPOA and the DBB occurs before sexual maturation of male rats.

Because polymorphisms in the methyl group metabolism genes methylene-tetrahydrofolate reductase (MTHFR), methionine synthase (MS), and cystathione beta-synthetase (CBS) affect plasma homocysteine levels and intracellular concentrations of S-adenosylmethionine (SAM), they modify the susceptibility to cardiovascular diseases and cancer. Specifically, genome-wide decreased DNA methylation ('hypomethylation') in human cancers might be a consequence of decreased SAM levels. Because hypomethylation is particularly prevalent in transitional cell carcinoma of the urinary bladder (TCC), the genotype distributions for the two each most prevalent MTHFR, MS, and CBS alleles were compared between 165 TCC patients and 150 population controls. The distributions of the MTHFR 677A/V and the MS 919G/D alleles were not significantly different between cancer patients and controls, even after stratification according to age, gender, tumor stage or grade. The CBS 844INS68 allele was slightly less frequent in TCC patients than in controls (q=0.07 versus 0.10), but was rarer among males in both groups. Among the TCC patients, this gender difference was highly significant (Mantel-Haenszel and chi(2)-test P=0.007). No significant difference between TCC patients and controls was found for any combined genotype. Likewise, the extent of DNA hypomethylation determined in 62 carcinoma specimens was not related to the respective genotypes. Thus, on their own, the MTHFR, MS and CBS genotypes do not appear to act upon susceptibility to TCC or influence the extent of DNA hypomethylation in this cancer.

The defibrinogenating effect of batroxobin (Defibrase) in male Wistar rats and the inhibitory effects of the plasma of batroxobin-treated rats on the migration of human vascular smooth muscle cells (SMCs) were investigated in vitro. At 1 h after a single intravenous injection of 3.0, 10.0 or 30.0 BU/kg batroxobin (ten rats in each group), the fibrinogen levels in the plasma of the rats decreased to 88.3, 66.2 and 16.5%, respectively, of that in the plasma of control saline-treated rats (261.0+/-26.7 mg/dl). When the plasma from the batroxobin-treated rats was added to Dulbecco's modified Eagle's medium at a concentration of 0.2% for a vascular SMC migration assay and incubated in a modified Boyden's chamber system at 37 degrees C for 24 h, significant inhibitory effects on vascular SMC migration were observed in the 10.0 (P<0.05) and 30.0 BU/kg (P<0.01) batroxobin-treated rats. The plasma of batroxobin-treated rats as well as standard rat fibrinogen induced vascular SMC migration in a fibrinogen content-dependent manner except the plasma of the 30.0 BU/kg batroxobin-treated rats. Moreover, the rat serum (0.1 approximately 5.0%) did not show any activity on vascular SMC migration in the present experimental system. These results indicate that the plasma fibrinogen significantly influences vascular SMC migration, and that the inhibitory effect of the plasma of batroxobin-treated rats on vascular SMC migration is related to the defibrinogenating action of batroxobin in vivo.

We examined effects of gap junction blockers, octanol and halothane, on circadian rhythms in the release of arginine-vasopressin (AVP) and vasoactive intestinal polypeptide (VIP) in suprachiasmatic nucleus (SCN) slice cultures of the rat. Circadian rhythms in AVP and VIP release maintained when the SCN culture was treated with octanol for 42 h. However, the release of AVP and VIP showed no circadian rhythms after 7 days incubation with octanol or halothane. Circadian rhythmicity in the two peptide rhythms appeared after the removal of the drug from the culture medium. These findings suggested that the gap junction communication may be involved in intercellular coupling within each subpopulation of AVP or VIP neurons in the SCN.

Wide innervation of cholinergic projections throughout the cortex implies that acetylcholine (ACh) plays an essential role in information processing, but how it works is still enigmatic. Experimental as well as theoretical work in the olfactory cortex and hippocampus suggests that ACh, via the muscarinic receptors, serves to shift the dynamics of the cortical networks into a state where afferent influence predominates over intracortical influence. Recent experiments in the visual and somatosensory cortex suggested that this hypothesis could be extended to neocortex. In addition, participation of the nicotinic receptors in regulating the synaptic response in the somatosensory cortex further substantiates this hypothesis. This hypothesis, derived mainly from in vitro work, also seemed to account for results from in vivo experiments without any obvious inconsistencies.

We examined two molecular responses to estrogen, reduction in estrogen receptor alpha (ER alpha) mRNA and increase in progesterone receptor (PR) mRNA, in the hypothalamus of 3- (young) and 10-month-old (middle-aged) cycling, and 15-month-old (old) acyclic, Fischer 344 female rats. The rats were ovariectomized and then given silastic capsules containing 5% 17beta-estradiol. or empty implants, and killed 4 days after implantation. By means of in situ hybridization, we found that, in young rats, estrogen reduced ER alpha mRNA in both the ventromedial hypothalamus (VMH) and arcuate nucleus (ARC) but not in the preoptic area (POA). In contrast, the effect of estrogen on ER alpha mRNA in the VMH and ARC of middle-aged and old rats was not statistically significant. On the other hand in all regions the induction of PR mRNA by estrogen was at least as strong in middle-aged and old as in young rats. The present study revealed that the induction of PR mRNA by estrogen in the hypothalamus was not impaired with age but ER alpha mRNA in the VMH and ARC was significantly impaired with age, but not in the POA.

CrkL is an adapter protein comprising Src homology (SH) 2 and SH3 domains. We investigated the molecule(s) associated with CrkL in factor-dependent cell lines. In the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent cell lines TF-1 and UT-7, an approximately 95-kDa tyrosine-phosphorylated protein was precipitated along with CrkL after GM-CSF stimulation. The same protein was also observed when we used the erythropoietin (EPO)-dependent cell line UT-7/EPO, in an EPO stimulation-dependent manner. We identified it as STAT5 (signal transducer and activator of transcription 5, 96 kDa) by STAT5-specific antibodies. The direct binding of the SH2 domain of CrkL to STAT5 was demonstrated in far Western blotting and pull-down experiments using the glutathione S-transferase (GST) fusion construct CrkL-SH2. The addition of the oligopeptide containing phosphotyrosine 694 in STAT5A impaired the association between GST-CrkL-SH2 and STAT5. Furthermore, in a gel shift assay using prolactin-inducible element (PIE) as the probe, the DNA binding activity of STAT5 was inhibited by the interaction with GST-CrkL-SH2 in vitro. Finally, we found that STAT5 associated with CrkL did not bind to PIE sequence. These results suggest that CrkL participates in the Janus kinase (JAK)-STAT pathway by direct association with STAT5., (Copyright 1998 Academic Press.)

5,9-Diacetamido-2,6-anhydro-O-4-carbamoylmethyl-3,5,9-trideo xy-D-glycero- D-galacto-non-2-enonic acid (1) was synthesized via a key intemediate 2 through the Neu5Ac aldolase [E.C.4.1.3.3]-catalyzed aldol reaction of 2-acetamido-2,6-dideoxy-6-azido-D-glucose with sodium pyruvate operating under alkaline conditions (pH 10.5) in order to accelerate epimerization C-2 of N-acetyl-D-glucosamine (D-GlcNAc) derivatives. Compound 1 showed inhibitory activity against sialidase.

We have isolated a novel cDNA encoding macrophage colony-stimulating factor (M-CSF) from a murine stromal cell line, ST2. The cDNA included an entire coding sequence of the M-CSF gene but contained an additional sequence of 140 base pairs (bp). Northern blot analysis demonstrated that other murine cell lines such as a fibroblastic cell line (L) and a stromal cell line (PA6) also expressed the transcripts corresponding to the clone. The nucleotide sequence analyses of the cDNA and the cloned M-CSF genome revealed that the 140-bp insertion sequence was part of intron 1 which separated exon 1 and exon 2: the former contained part of the amino acid residues of the signal sequence and the latter the rest of the signal sequence and the first 22 amino acid residues of the mature protein. The insertion of the 140-bp intron sequence not only changed the amino acid sequence of the signal peptide but also generated an in-frame termination codon. However, instead of the dysfunction of the original initiation codon, the 140-bp insertion sequence contained a putative ATG initiation codon that preserved the original open reading frame. Finally, we found that the cDNA directed the expression of a secreted and biologically active M-CSF protein when it was introduced into COS7 cells and M-CSF activity in the culture supernatants was measured using an M-CSF-dependent cell line. These results indicate the presence of an alternatively spliced M-CSF transcript which utilizes an alternate initiation codon in order to specify active M-CSF protein., (Copyright 1998 Academic Press.)