Ingestion of paddy rice increases intestinal mucin secretion and goblet cell number and prevents dextran sodium sulfate-induced intestinal barrier defect in chickens.

Paddy rice is a potential feed grain for chickens, whose strong gizzards can crush the hull. Here, we investigated whether paddy rice rich in hull-derived water-insoluble dietary fiber stimulates intestinal mucin secretion and production, as well as the possible involvement of paddy rice in intestinal barrier function. Layer male chicks at 7 d of age were divided into four groups according to the diet: corn, polished rice, brown rice, or paddy rice (650 g/kg diet), which they ate for 14 consecutive days. At 21 d of age, the birds were refed their experimental diets, and small intestinal mucin fractions were collected to determine intestinal mucin content. Small intestinal mucin secretion was induced most strongly in the paddy rice group (Experiment 1). The rank order of diet-induced mucin secretion was paddy rice > corn = brown rice > polished rice. Ileal MUC2 gene expression and ileal number of goblet cells were highest in the paddy rice group (Experiment 1). A study of bromodeoxy-U uptake into ileal epithelial cells indicated the increase in goblet cells in the paddy rice group was related to accelerate epithelial cell migration (Experiment 2). A single supplementation of isolated rice hulls without kernels increased MUC2 gene expression and goblet cell numbers (Experiment 3), suggesting the importance of the hull's bulk-forming capacity on mucin production. Finally, chicks fed corn or paddy rice were orally administered dextran sodium sulfate (DSS) to disrupt intestinal barrier function. In the DSS-treated birds, the intestinal permeability of fluorescein isothiocyanate dextran in the everted gut sacs was much lower in the paddy rice group than in the corn group (Experiment 4), showing that paddy rice protects against mucosal disruption. In conclusion, ingestion of paddy rice increases intestinal mucin secretion and production through enhanced MUC2 gene expression and epithelial turnover and prevents DSS-induced intestinal barrier defects in chickens.

Rapid incorporation of carbon-11-labeled diacylglycerol as a probe of signal transduction in glioma.

We have synthesized and characterized a positron-emitting carbon-11-labeled 1,2-diacylglycerol to study phosphoinositide turnover in tumor cells. Rapid incorporation of the 1,2-diacylglycerol was observed in the C6 glioma cell line. The incorporated lipid fraction consisted chiefly of phosphoinositide pool and another phospholipid pool in the proliferative state. When the state was inhibited by (-)-3D-3-deoxy-3-fluoro-myo-inositol, incorporation into the phosphoinositide pool decreased selectively. This suggested that phosphoinositide turnover is the leading regulator of tumor proliferation potential. On the basis of the concept of carbon-11-labeled 1,2-diacylglycerol as a specific probe for visualizing the tumor signal transduction in vivo, we obtained proliferating images of implanted C6 glioma cells in the rat brain by autoradiography and visualized the proliferation signal in human glioma by positron emission tomography.

Functional domains of c-Myc involved in the commitment and differentiation of murine erythroleukemia cells.

In the early event of the induction of mouse erythroleukemia (MEL) cell differentiation, c-myc mRNA levels show a drastic change. The elevated expression of a transfected c-myc gene inhibits the commitment and differentiation of MEL cell transformants. In the present work, we have introduced human c-myc mutants into MEL cells under the inducible promoter to define the functional domains of c-Myc involved in erythroid differentiation. The c-Myc domains necessary for commitment and differentiation are not co-localized; almost entire regions are required for inhibition of commitment, whereas domains II and IV that are essential for co-transforming activity with ras are required for inhibition of differentiation. Interestingly, mutants that delete domains for c-Myc dimerization motifs enhanced differentiation. These results suggest that c-Myc may regulate commitment and differentiation by interacting with proteins through different domains.

Effects of recombinant human interleukin 1 alpha and interleukin 1 beta on cell growth and alkaline phosphatase of the mouse osteoblastic cell line MC3T3-E1.

Recombinant human interleukin 1 (rhIL-1)alpha and rhIL-1 beta were examined for their effects on DNA synthesis, cell growth and alkaline phosphatase activity of the mouse osteoblastic cell line MC3T3-E1. The relative activity of rhIL-1 alpha and rhIL-1 beta was compared in terms of the units which induced half-maximal [3H]thymidine uptake into mouse thymocyte cultures exposed to IL-1. Both rhIL-1 alpha and rhIL-1 beta significantly inhibited DNA synthesis and division of the cells in a concentration- and cultivation time-dependent fashion. In contrast, rhIL-1 alpha and rhIL-1 beta markedly increased alkaline phosphatase activity, which is a marker of osteoblastic differentiation. This activity in cells treated with rhIL-1 alpha and rhIL-1 beta increased about 2.0- and 1.7-fold, respectively, compared with that of control cultures. Inhibition of the DNA synthesis and stimulation of alkaline phosphatase activity by both types of rhIL-1 were completely neutralized by treatment with their respective polyclonal antisera. Also, inhibition of DNA synthesis was unaffected by the addition of cyclooxygenase and lipoxygenase inhibitors, and stimulation of alkaline phosphatase activity was unaffected by the addition of indomethacin. These results indicate that both rhIL-1 alpha and rhIL-1 beta have qualitatively similar biological effects on osteoblastic cells. They also suggest that IL-1 is an important modulator of the growth and differentiation of osteoblasts.

Regulation of macrophage gene expression by T-cell-derived lymphokines.

Cytokines secreted from antigen-specific T lymphocytes provide important positive and negative control of inflammation through their effects on non-antigen-specific inflammatory leukocytes. These effects often involve modulation of gene expression. Lymphokine-inducible macrophage gene expression is largely controlled at the level of transcription. Multiple cis-acting sequence motifs cooperate with one another to produce patterns of expression that are relatively unique to individual genes. Members of trans-acting transcription factor families, which recognize related regulatory sequence elements, participate frequently in complex protein-protein interactions that generate remarkable complexity in terms of the number of potential combinations and the consequential functional differences exhibited by each combination. Thus, the remarkable plasticity of immune-mediated inflammation derives from combinations of finite numbers of options at several points in the cellular and molecular sequence.

Positron emission tomography-based boron neutron capture therapy using boronophenylalanine for high-grade gliomas: part I.

Determination of tumor boron-10 (10B) levels is required for accurate neutron dosimetry during boron neutron capture therapy. We assessed a new method for quantitative measurement of boronated drug uptake in high-grade gliomas. This method uses positron emission tomography (PET) with fluorine-18-labeled L-fluoroborono-phenylalanine (L-18F-10B-FBPA), which was synthesized as an analogue of L-boronophenylalanine. We studied the accumulation of L-18F-10B-FBPA by PET in patients with high-grade gliomas. Dynamic PET studies of brain tumors revealed that L-18F-10B-FBPA accumulated gradually after bolus injection, and the value of PET activity divided by the integrated plasma activity reached a constant level 42 min after injection, which was defined as the incorporation constant (Ic*). This constant reflected the appropriate L-18F-10B-FBPA accumulation in tumor tissue. Based on the Ic* constant, the methods for estimating tumor 10B concentration were devised. With this method, the estimated values of 10B concentration in gliomas were very close to the 10B levels in surgical specimens. This method was based solely on PET and can potentially provide data that would assist in the selection of patients for future treatment with boron neutron capture therapy after surgical resection of their brain tumors.

Positron emission tomography-based boron neutron capture therapy using boronophenylalanine for high-grade gliomas: part II.

Based on pharmacokinetic findings of fluorine-18-labeled L-fluoroboronophenylalanine by positron emission tomography (PET), methods for estimating tumor 10B concentration were devised. In clinical practice of boron neutron capture therapy (BNCT) for high-grade gliomas, a large amount of L-boronophenylalanine (L-10B-BPA)-fructose solution is used. Under these conditions, a slow i.v. infusion of L-10B-BPA-fructose solution should be performed for BNCT; therefore, the changes over time in 10B concentration in the target tissue were estimated by convoluting the actual time course of changes in plasma 10B concentration with a PET-based weight function including the proper rate constants [K1 (ml/g/min), k2 (min(-1)), k3 (min(-1)), and k4 (min(-1))]. With this method, the estimated values of 10B concentration in gliomas were very close to the 10B levels in surgical specimens. This demonstrated the similarity in pharmacokinetics between fluorine-18-labeled L-fluoroboronophenylalanine and L-10B-BPA. This method, using the appropriate rate constant, permits the determination of tumor 10B concentration and is widely suitable for clinical BNCT, because the averaged PET data are enough to use in future patients without individual PET study.

Requirement for STAT1 in LPS-induced gene expression in macrophages.

This study examines the role of the signal transducer and activator of transcription 1 (STAT1) in induction of lipopolysaccharide (LPS)-stimulated gene expression both in vitro and in vivo. LPS-induced expression of an interferon (IFN)-inducible 10-kDa protein (IP-10), IFN regulatory factor-1 (IRF-1), and inducible nitric oxide synthase (iNOS) mRNAs was severely impaired in macrophages prepared from Stat1-/- mice, whereas levels of tumor necrosis factor alpha and KC (a C-X-C chemokine) mRNA in LPS-treated cell cultures were unaffected. A similar deficiency in LPS-induced gene expression was observed in livers and spleens from Stat1-/- mice. The reduced LPS-stimulated gene expression seen in Stat1-/- macrophages was not the result of reduced activation of nuclear factor kappaB. LPS stimulated the delayed activation of both IFN-stimulated response element and IFN-gamma-activated sequence binding activity in macrophages from wild-type mice. Activation of these STAT1-containing transcription factors was mediated by the intermediate induction of type I IFNs, since the LPS-induced IP-10, IRF-1, and iNOS mRNA expression was markedly reduced in macrophages from IFN-alpha/betaR-/- mice and blocked by cotreatment with antibodies against type I IFN. These results indicate that indirect activation of STAT1 by LPS-induced type I IFN participates in promoting optimal expression of LPS-inducible genes, and they suggest that STAT1 may play a critical role in innate immunity against gram-negative bacterial infection.

A lipopolysaccharide-inducible macrophage gene (D3) is a new member of an interferon-inducible gene cluster and is selectively expressed in mononuclear phagocytes.

We previously reported the isolation and characterization of cDNA clones encoding novel lipopolysaccharide (LPS)-inducible mRNAs from murine peritoneal macrophages. We now present the complete coding sequence of a cDNA previously termed D3. Analysis of multiple clones from a murine macrophage cDNA library provided a complete cDNA sequence of approximately 1.6 kb. The corresponding RNA contains a single open reading frame encoding a hydrophilic protein composed of 425 amino acids and is characterized by a region including three perfect and two imperfect repeats of a seven-amino-acid sequence. Based on nucleotide and deduced amino acid sequence, this mRNA is a new member of a previously described multigene cluster of interferon-inducible genes termed the Mouse 200 series genes. This new sequence most closely resembles gene 204 because both D3 and 204 genes have segments containing the seven-amino-acid repeat sequence. The Mouse 202 and 204 genes, however, have an approximately 200-amino-acid carboxyl-terminal domain that is absent in the LPS-inducible macrophage-derived cDNA. In addition, D3, 202, and 204 can all be distinguished from one another by virtue of unique 3' noncoding regions 200-300 base pairs in length. The D3 unique sequence is largely restricted to the smallest of the three size classes of this gene family expressed in macrophages and is not detected in interferon- or platelet-derived growth factor-stimulated fibroblasts. Overall, three separate mRNAs have now been described, each of which has three or more of a possible seven nucleotide sequence domains. Although the function(s) of the members of this gene family remains unknown, the multiple forms inducible by diverse stimuli and their restricted cell type expression suggest diverse and important physiologic roles for their products in inflammation.

Human purified interleukin-1 inhibits DNA synthesis and cell growth of osteoblastic cell line (MC3T3-E1), but enhances alkaline phosphatase activity in the cells.

We examined the effects of human purified interleukin-1 (IL-1) on DNA synthesis, cell growth, and alkaline phosphatase activity in the osteoblastic cell line MC3T3-E1 under both preconfluent and confluent culture conditions. Addition of IL-1 to the cells markedly inhibited their DNA synthesis and growth over the range 1-10 U/ml. Such significant inhibitory effects were observed in cells cultivated in 1 or 5% fetal calf serum (FCS)-containing alpha modification Eagle's medium (alpha-MEM), but not in alpha-MEM containing 10% FCS. In contrast, alkaline phosphatase activity was enhanced significantly by IL-1 in the cell line cultivated in 1% FCS-containing alpha-MEM. These results demonstrate that human purified IL-1 is effective in inducing the differentiation of osteoblastic cell MC3T3-E1.

Location of motoneurons supplying upper neck muscles in the chicken studied by means of horseradish peroxidase.

The distribution of motoneurons innervating the upper cervical muscles, biventer cervicis, splenius capitis, complexus, rectus capitis dorsalis, rectus capitis lateralis, and rectus capitis ventralis in the chicken was examined by retrograde transport of horseradish peroxidase. Labeled motoneurons supplying upper neck muscles ranged from 30 to 60 micron in diameter and were located within two subnuclei in the brainstem. The rostrocaudal distributions of motoneurons projecting to individual cervical muscles ranged from 3 to 9 mm in length, both rostral and caudal to the obex. Detailed analysis of the data showed that the more dorsally positioned subnucleus projected mainly to the hypaxial muscles, i.e., the rectus capitis ventralis and lateralis, whereas the ventral subnucleus supplied chiefly the epaxial muscles, i.e., the biventer cervicis and splenius capitis. The complexus and rectus capitis dorsalis were innervated by both of these subnuclei. Historically these dorsal and ventral subnuclei, respectively, have been called the nucleus hypoglossus ventralis and the nucleus hypoglossus ventralis ventrolateralis. In view of the observation that these nuclei do not undergo retrograde degeneration following section of the hypoglossal nerves, this older nomenclature is misleading. In agreement with other authors, we suggest that these motoneuron groups should be collectively referred to as the nucleus supraspinalis.

The effects of pravastatin on hyperlipidemia in renal transplant recipients.

Hyperlipidemia may be one of the risk factors in the development of atherosclerotic disease in renal transplant recipients. In the present study, 24 kidney recipients with hyperlipidemia were treated with an HMG-CoA reductase inhibitor, pravastatin (10 mg/day). All recipients had been treated with cyclosporine (CsA), azathioprine (Az), and prednisolone (Pred). The mean total cholesterol (T-chol) level decreased from 323 +/- 7.4 to 261 +/- 7.9 mg/dl at one month after starting treatment (P less than 0.01) and this level did not change during treatment for further 6 months. The mean LDL cholesterol level was also decreased from 205.9 +/- 11.2 to 118.7 +/- 8.1 mg/dl at 3 months after starting treatment (P less than 0.01). On the other hand, pravastatin did not affect the levels of HDL-cholesterol and triglycerides. Pravastatin did not show any effects on the white blood cell, monocyte, and lymphocyte counts, or the hemoglobin concentration (NS). One patient displayed a slight elevation of aspartate aminotransferase and alanine aminotransferase levels, but this was not sufficient to cease treatment. Pravastatin did not adversely affect the renal function or creatinine phosphokinase (CPK) levels. Two recipients developed nausea and vomiting and their treatment was stopped. Pravastatin appears to be a safe and efficacious method of treating hyperlipidemia in renal transplant recipients.

Spontaneous regression of intracranial arteriovenous malformation on PET.

We report a case of spontaneous regression of intracranial arteriovenous malformation (AVM) detected by PET in a 57-yr-old woman who had suffered repeated ruptures of the AVM at 28, 30 and 31 yr of age associated with pregnancy. The rupture at this hospitalization was the most critical, and after repeated ruptures for 1 mo, the AVM regressed spontaneously. The decreased cerebral blood volume (CBV) in the AVM indicated regression. The flow-to-volume (CBF/ CBV) ratios surrounding the AVM increased. The metabolic rate for oxygen (CMRO2) did not correlate with her improved neurological status, and an imbalance between CBF and CMRO2 was recognized.

Phosphoinositide turnover imaging linked to muscarinic cholinergic receptor in the central nervous system by positron emission tomography.

Receptor-mediated membrane processing plays an essential role in neural function in the synapses. In such neurotransmission process, the phosphoinositide (PI) response, an effector in the production of second-messengers, can be used to assess in vivo signal transduction. Using in vivo autoradiography and positron emission tomography (PET), we attempted to visualize the PI response to muscarinic cholinergic receptor (mAChR)-stimulation in rats and monkeys, which were administered 1,2-[11C]diacylglycerol (DAG) intravenously. Enhancement of 1,2-[11C]DAG incorporation was observed in the rat ipsilateral hippocampus and cortex in which mAChR-agonist was administered by local injection, but this was in contrast to spreading cortical depression in the ipsilateral cortex using KCl. In monkey PET studies, dynamic brain scanning revealed increase in activity over time for about 15 min after a bolus injection of 1,2-[11C]DAG in an awake state. The activity then remained at a constant level. This finding documented the theoretical "membrane-trapping" mechanism. The systemic mAChR-stimulation accelerated incorporation in the cerebral cortices of the same monkey brain. Radioactivity uptake did not differ significantly between the mAChR-stimulated and nonstimulated early scan images. This suggested that cerebral blood flow does not greatly affect DAG incorporation. In sequential membrane processes of PI turnover, diacylglycerol kinase rapidly metabolizes DAG, included in PI turnover. In conclusion 1,2-[11C]DAG incorporation was limited by receptor-mediated PI turnover, which can represent real synaptic transmission in neural networks.

Protein kinase C imaging using carbon-11-labeled phorbol esters: 12-deoxyphorbol 13-isobutyrate-20-[1-11C]butyrate as the potential ligand for positron emission tomography.

Protein kinase C plays a crucial role in signal transduction for a variety of biologically active substances which activates cellular functions and their proliferation. The actions are closely related to both normal and abnormal functions in the nervous system. Tumor-promoting phorbol esters can substitute for diacylglycerols which are important ligands that bind to protein kinase C. Three typical phorbol esters, phorbol 13-[1-11C]butyrate, phorbol 12,13-[1-11C]dibutyrate and 12-deoxyphorbol 13-isobutyrate-20-[1-11C]butyrate, were synthesized by using [11C]ethylketene with a high specific activity (186GBq/mumol). Their in vivo autoradiograms demonstrated a heterogenous distribution in rat brain. 12-deoxyphorbol 13-isobutyrate-20-[1-11C]butyrate was particularly suited for in vivo use due to its nontumor-promoting activity and its ready permeability to the blood-brain barrier. High optical density was observed in the cortex, amygdala and hippocampus. The in vivo binding properties of this compound to protein kinase C were confirmed by in vivo displacement studies with unlabeled 12-deoxyphorbol 13-isobutyrate-20-butyrate and unlabeled phorbol 12,13-dibutyrate. This suggests that 12-deoxyphorbol 13-isobutyrate-20-[1-11C] butyrate has a specific binding affinity for protein kinase C.

Cerebral hemodynamics and metabolism of severe diffuse brain injury measured by PET.

Cerebral hemodynamics and metabolism in three patients with severe diffuse brain injury were measured 10 days after onset using PET. In this study, regional cerebral blood flow (rCBF), oxygen extraction fraction (rOEF), cerebral blood volume (rCBV), cerebral metabolic rate for oxygen (rCMRO2), cerebral metabolic rate for glucose (rCMRglc) and cerebral metabolic ratio (rCMRO2/rCMRglc) were measured. The Glasgow Coma Scale scores on admission were 4, 5 and 5, respectively, and CT on admission showed typical findings of diffuse brain injury. As a result, PET revealed misery perfusion and hyperglycolysis in Patient 1 and matching low perfusion and low glucose metabolism in Patients 2 and 3. Although Patient 1 died, Patients 2 and 3 had good recoveries. We speculate that a long-lasting anaerobic state, indicated by a high OEF value and low metabolic ratio, is an important undesirable factor related to the outcome.

Fluorine-18-labeled fluoroboronophenylalanine PET in patients with glioma.

UNLABELLED: We synthesized fluorine-18-labeled fluoroboronophenylalanine (18F-10B-FBPA), an analog of boronophenylalanine (10B-BPA), and characterized its pharmacokinetics in patients with glioma. We conducted PET studies on three types of gliomas to clarify the relationship between tumor grade and each rate constant [K1 (ml/g/min), k2 (min[-1]) and k3 (min[-1])], and here, we discuss the metabolism of the 10B-BPA analog (18F-10B-FBPA). METHODS: Thirty-three cases of primary gliomas were studied by dynamic PET using DL-18F-10B-FBPA or L-18F-10B-FBPA. Dynamic PET images of 18F-10B-FBPA incorporation into tumors were obtained, and the arterial blood samplings were performed in all cases. RESULTS: When the dynamic PET data were represented as Gjedde-Patlak plots, there was a positive slope, suggesting the involvement of the putative metabolic pool of this tracer. A three-compartment model using rate constants (K1, k2 and k3) was used for the kinetic analysis. The accumulation of 18F-10B-FBPA was found to correlate with the degree of malignancy, and the L form of 18F-10B-FBPA was taken up better than was the DL form. The results of dynamic PET analysis suggested that K1 (measuring amino acid transport process) is a major factor determining the accumulation of 18F-10B-FBPA. A comparison of the rate constants revealed that k3 (metabolic process) did not correlate with the degree of malignancy. The absence of evident differences in k3 between DL and L forms suggests that k3 represents phenomena that are not dependent on the native form of L. CONCLUSION: These PET data will be of practical use for diagnosis of malignancy and direct prediction of the effectiveness of boron neutron capture therapy using 10B-BPA.

Evaluation of phosphoinositide turnover on ischemic human brain with 1-[1-11C]-butyryl-2-palmitoyl-rac-glycerol using PET.

UNLABELLED: It is important to evaluate cerebral function from neural signal transduction in ischemic brain in judging morbid state and prognosis. We synthesized 1-[1-(11)C]-butyryl-2-palmitoyl-rac-glycerol (DAG) for the purpose of imaging the second messenger on PET and applied it to clinical cases of cerebral infarction. METHODS: Five patients, who had ischemic stroke, were examined with PET. [15O]-CO2 and [15O]-O2 inhalation methods were applied to cerebral blood flow (CBF), oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2). For the measurement of phosphoinositide turnover after intravenous injection of DAG, dynamic PET data were collected continuously for 46 min. Arterial blood samples were taken to evaluate changes in the serum concentration of DAG. To quantify the metabolic activity of inositol phospholipid, the incorporation constant k*(DAG) was calculated on the basis of the kinetics of DAG. RESULTS: The plasma concentration of DAG increased rapidly and peaked 30 s after injection of DAG solution. In the normal cortex, DAG concentration increased gradually and reached a plateau between 15 and 20 min after injection. In the ischemic core (infarction), DAG concentration increased slowly, and its peak concentration was lower than that in normal tissue. In comparison with blood flow and metabolic parameters, k*(DAG) showed the best correlation with CMRO2, suggesting a reflection of neuronal activity. Locally, CBF and CMRO2 gradually decreased from the normal area toward the ischemic center (infarction), whereas k*(DAG) and OEF significantly decreased only in the ischemic center. CONCLUSION: The k*(DAG) of ischemic brain, including that caused by infarction, significantly correlated with CMRO2, suggesting that metabolic activity of inositol phospholipid reflects neural viability. Maintained metabolic activity of inositol phospholipid in the region around the ischemic core indicated preservation of the signal transduction system through the metabotropic receptor.

Prosthetic replacement of entire left hemidiaphragm in malignant fibrous histiocytoma of the diaphragm.

A case of primary malignant fibrous histiocytoma of the diaphragm which, to our knowledge, is the first record in the world literature, is presented. It occupied almost the entire left hemidiaphragm and was surgically removed with the left lower pulmonary lobe, stomach, colon, and spleen. The entire left hemidiaphragm then was replaced with Marlex mesh. Prosthetic replacement of the entire hemidiaphragm, which has not been previously reported, was successfully performed with preservation of pulmonary function on the affected side. The patient died of brain metastases of malignant fibrous histiocytoma more than 4 months postoperatively. Diagnosis of diaphragmatic tumors and a technique which involves the use of a prosthesis are discussed.