Phosphorylation of Smad2/3 at the specific linker threonine residue indicates slow-cycling esophageal stem-like cells before re-entry to the cell cycle.

UNLABELLED: The stem cell compartment in the esophageal epithelium is possibly located in the basal layer. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in the epithelial cells of murine stomach and intestine, and have suggested that these cells are epithelial stem cells. In this study, we explore whether pSmad2/3L-Thr could serve as a biomarker for esophageal stem cells. We examined esophageal tissues from normal C57BL/6 mice and those with esophagitis. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, CDK4, p63, or CK14 was performed. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. We used the 5-bromo-2-deoxyuridine (BrdU) labeling assay to examine label retention of pSmad2/3L-Thr immunostaining-positive cells. We collected specimens 5, 10, 15 and 20 days after repeated BrdU administrations and observed double immunofluorescent staining of pSmad2/3L-Thr with BrdU. In the esophagus, pSmad2/3L-Thr immunostaining-positive cells were detected in the basal layer. These cells were detected between Ki67 immunostaining-positive cells, but they were not co-localized with Ki67. pSmad2/3L-Thr immunostaining-positive cells showed co-localization with CDK4, p63, and CK14. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features. Until 20 days follow-up period, pSmad2/3L-Thr immunostaining-positive cells were co-localized with BrdU. pSmad2/3L-Thr immunostaining-positive cells significantly increased in the regeneration phase of esophagitis mucosae, as compared with control mice (esophagitis vs. CONTROL: 6.889 ± 0.676/cm vs. 4.293 ± 0.659/cm; P < 0.001). We have identified significant expression of pSmad2/3L-Thr in the specific epithelial cells of murine esophagi. We suggest that these cells are slow-cycling epithelial stem-like cells before re-entry to the cell cycle.

Expression of Forssman antigen synthesis and degradation in human lung cancer.

The activities of the two enzymes (UDP-acetylgalactosamine-globoside alpha-N-acetylgalactosaminyltransferase and alpha-N-acetyl-D-galactosaminidase) involved in the synthesis and degradation of Forssman antigen were studied in uninvolved and neoplastic human lungs. The Forssman synthetic enzyme activities of 17 of 18 squamous cell carcinomas were higher than those of the uninvolved lung tissues of the subjects studied, and the degradation enzyme activities of 16 of 18 squamous cell carcinomas were higher than those of the uninvolved portions. No consistent abnormalities in both enzyme activities were seen in 28 adenocarcinomas, whereas the mean activities of the two enzymes were elevated in these neoplasms. These differences in enzyme activities between those samples may indicate that the synthesis and degradation of Forssman antigen in adenocarcinoma of the lung are expressed or repressed according to the individual, whereas in squamous cell carcinoma, these activities are expressed unrelated to the individual.

Cyclic AMP inhibits the respiratory burst of electropermeabilized human neutrophils at a downstream site of protein kinase C.

We studied a signaling pathway for the activation of the superoxide (O2-)-generating NADPH oxidase and effects of cAMP on the pathway using electropermeabilized human neutrophils. The permeabilized cells produced O2- by the addition of protein kinase C (PKC) activator, phorbol myristate acetate (PMA), and a non-hydrolyzable GTP analogue, GTP gamma S in the presence of ATP and Mg2+. The O2- production by PMA not by GTP gamma S was inhibited by inhibitors of PKC. The production by PMA and GTP gamma S was inhibited by a GDP analogue, GDP beta S, in the same dose-dependent manner and the production by PMA was not enhanced by the addition of GTP gamma S and vice versa. These findings suggest the presence of a GTP-binding protein which follows PKC in the activation pathway. The O2- production by PMA and GTP gamma S was dose-dependently inhibited by cAMP and the inhibition was completely restored by an inhibitor of cAMP-dependent protein kinase, H-89, indicating that cAMP blocks the activating pathway at the site between the GTP-binding protein located downstream of PKC and the NADPH oxidase by activating cAMP-dependent protein kinase. The activation of the oxidase by sodium dodecyl sulfate (SDS) seemed to be different from the above pathway. It needed higher concentrations of GDP beta S for inhibition, did not absolutely need ATP and was inhibited by neither cAMP nor protein kinase C inhibitors. Moreover, the O2- production by the combination of GTP gamma S and SDS or of PMA and SDS was essentially the same as the sum of the production by each stimulant alone. We may conclude from the observations that the signaling pathway involving PKC for the activation of the oxidase is distinct from the pathway induced by SDS: the former is blocked by cAMP at the site between the GTP-binding protein located downstream of PKC and the oxidase and the latter is cAMP-insensitive.

Phosphatidic acid induces the respiratory burst of electropermeabilized human neutrophils by acting on a downstream step of protein kinase C.

Phosphatidic acid (PA) dose-dependently induced superoxide (O2-) production of electropermeabilized human neutrophils but not of intact neutrophils, indicating that PA induces the activation of NADPH oxidase by acting on an intracellular target. The O2- production by PA was not inhibited by protein kinase C (PKC) inhibitors, such as staurosporine and calphostin C, and an inhibitor of PA phosphohydrolase, propranolol. These observations suggest that the activation of the oxidase by PA is independent of the activity of PKC and may dominate the activation by diacylglycerol which is formed from PA via the action of PA phosphohydrolase. Furthermore, the production by PA, as well as that by phorbol myristate acetate, was inhibited by cyclic AMP and GDP beta S. Therefore, PA seems to act at a site downstream of PKC.

Tyrosine phosphorylation is involved in the respiratory burst of electropermeabilized human neutrophils at a step before diacylglycerol formation by phospholipase C.

We studied a step where tyrosine phosphorylation is involved in a signaling pathway for the activation of the superoxide (O2-)-generating NADPH oxidase using electropermeabilized human neutrophils. The permeabilized cells produced O2- by the addition of a protein tyrosine phosphatase inhibitor, vanadate, as well as N-formyl-methionyl-leucyl-phenylalanine (fMLP) and protein kinase C (PKC) activators such as phorbol myristate acetate (PMA) and L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol (OAG). The O2- production by the stimulants was completely inhibited by PKC inhibitors such as calphostin C and staurosporine and was not affected by 1% ethanol, a metabolic modulator of phospholipase D (PLD). Furthermore, the O2- production by vanadate and fMLP, but not by OAG and PMA, was inhibited by both an inhibitor of phospholipase C (PLC), neomycin, and an inhibitor of tyrosine kinase, ST-638. These findings suggest that tyrosine phosphorylation is involved in the activation of the oxidase at a step before diacylglycerol formation by PLC, and that PLD may not be involved in the signaling pathway in permeabilized cells.

Purification and properties of galactosylceramide sulfatase activator from human liver.

Activator protein for galactosylceramide sulfatase (GSase) was purified from human liver. The activator has an approximate molecular weight of 22,000, is glycoprotein in nature, and is most probably a trimer consisting of an 8,000 dalton monomer. Monospecific rabbit antiserum raised against the activator strongly inhibited the activity of the activator. In the presence of a 10-fold or more excess of galactosylceramide sulfate (GS) on a molar basis, GS binding to the GSase activator occurred, and was saturated at an equimolar ratio. Binding studies on the GSase activator were conducted using affinity chromatography on derivatives of GS as ligands, and gel filtration of mixtures containing glycolipids and the activator. A "GS-acid" derivative, which was prepared by oxidative cleavage of sphingosine moiety in GS, and a sulfonamide derivative of GS as ligands still retained affinity for the GSase activator, while a hydrophobic ligands, an aminohexyl group did not bind completely the activator. A ligand of "galactosylceramide-acid" had weak affinity for GSase activator. These results suggest that the sulfate group and one of the two hydrocarbon chains in GS are not essential for the binding of the activator. The affinity of galactosylceramide for the GSase activator was confirmed by the detection of the lipid-protein complex on gel filtration. The activator weakly stimulated porcine GM1-beta-galactosidase activity.

Phosphatidic acid induces the release of beta-glucuronidase but not lactoferrin from electropermeabilized human neutrophils.

We studied the degranulation reaction of electropermeabilized human neutrophils induced by 1,2-didecanoyl-3-sn-phosphatidic acid (PA10). PA10 dose-dependently induced the release of beta-glucuronidase, an enzyme of azurophil granules, but did not induce the release of lactoferrin, a protein of specific granules. The enzyme release by PA10 absolutely required Ca2+, ATP, and Mg2+ and the concentrations for the half-maximal response were 2.5 microM, 60 microM, and 0.25 mM, respectively. Although Ca2+ alone at concentrations higher than 10 microM induced the release of both beta-glucuronidase and lactoferrin, the extents of the release were far less than that of the beta-glucuronidase release by PA10. Phorbol myristate acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol induced the release of lactoferrin alone at concentrations of Ca2+ below 0.5 microM while they induced the release of both beta-glucuronidase and lactoferrin at higher Ca2+ concentrations, indicating that the degranulation induced by PA10 is not mediated by diacylglycerol which might be formed from PA. The degranulation reactions induced by PA10 and PMA were dose-dependently inhibited by staurosporine and calphostin C, protein kinase C inhibitors, although no direct activation of protein kinase C by PA10 was observed. The extent of the beta-glucuronidase release by PA10 was not enhanced by the addition of PMA. Propranolol, which inhibits protein kinase C as well as phosphatidic acid phosphohydrolase, strongly inhibited the degranulation reactions induced by PA10 and PMA. Ethanol, a metabolic modulator of phospholipase D, and cyclic AMP did not affect the degranulation reactions by PMA and PA10.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of the effects of steroids on experimental septic lung injury.

To evaluate the clinical usefulness of steroids for septic lung injury, we investigated the effects of methylprednisolone (MP) on this disorder using an experimental rat model of cecal ligation and puncture (CLP). While 92% of the rats that underwent CLP (CLP rats) died within 30 h, those given high-dose MP (30 mg/kg) just after the operation (CLP + MP rats) survived for a significantly longer period (p < 0.01). Concentrations of endotoxin (ET) in arterial blood were significantly higher in the CLP + MP rats than in the CLP rats, while those in the bronchoalveolar lavage fluid (BALF) were significantly lower. Alveolar macrophages (AM) obtained from the CLP rats (CLP-AM) generated more O2-than did AM from sham-operated rats (sham-AM) following stimulation. However, the administration of MP did not reduce the upregulated generation of O2-by CLP-AM. While CLP-AM produced less leukotriene (LT)B4 than did sham-AM following stimulation with A23187, the administration of MP further reduced LTB4 production. When AM were cultured with [3H]arachidonic acid (3H-AA), the uptake of the isotope and the 3H release were significantly less in CLP-AM than in sham-AM. The administration of MP did not cause recoveries in the uptake and release of 3H-AA by CLP-AM. Although the survival time of CLP rats was significantly prolonged and the translocation of ET into BALF was reduced by steroid administration, the steroid effects were not explained by those on altered AM function. The upregulated generation of O2- and reduced LTB4 production from CLP-AM were not reversed by the treatment of this drug.

Prenatal diagnosis of fucosidosis.

Pregnancy from a family at risk for fucosidosis was monitored. The fetus was diagnosed as having a carrier state of the disease. alpha-L-fucosidase activity, however, was found to be absent in white blood cells obtained from identical twins after delivery. The diagnostic evaluation of (1) the enzyme activity in amniotic fluid and in cultivated amniotic fluid cells and (2) the presence of fucose rich compound in amniotic fluid are discussed.

Glucose-6-phosphatase activity in liver and blood platelets of two patients with glycogen storage disease type I.

Glucose-6-phosphatase (G-6-Pase) activity in liver and blood platelets of two patients with glycogen storage disease (GSD) type I is described. Both patients had a reduced activity of G-6-Pase in liver. The km value for glucose 6-phosphate (G-6-P) of residual activity in liver of both patients was similar to that of control liver. We could not demonstrate any reduced activity of platelet G-6-Pase in the patients. Platelet G-6-Pase with our assay method seems to represent a nonspecific phosphatase activity. Our observation suggests that it is necessary to examine platelet G-6-Pase of many other patients with GSD type I to confirm that G-6-Pase deficiency can be diagnosed by enzyme assay performed on blood platelets.

Diaphragm pacing with the spinal cord stimulator.

BACKGROUND: Diaphragm pacing with electrical stimulation of the phrenic nerve is an established treatment for central hypoventilation syndrome. The device, however, is not readily available, at least, in Japan. We applied the spinal cord stimulator for pain control to phrenic nerve stimulation. The purpose of this study is to evaluate the efficacy and feasibility of phrenic pacing with the compromise method. METHOD AND PATIENTS: We implanted a spinal cord stimulator in five patients with chronic central hypoventilation. The stimulation electrode was placed along the phrenic nerve in the neck, and the device was implanted in the anterior chest. We used the cyclic mode, and set the parameters at 1 second ramp up, 2 seconds on, 3 seconds off. The pulse width and the frequency were set at 150 microsec and 21 Hz respectively. The amplitude was adjusted to obtain sufficient tidal volume and to maintain PaCO2 at around 40 mmHg. FINDINGS: During the follow-up period from 3 to 34 months (mean 23), stable and sufficient ventilation were observed in all patients without complications. INTERPRETATION: Although longer follow-up is necessary, diaphragm pacing with the spinal cord stimulator is feasible for treatment of central hypoventilation syndrome.

Hyperresponsiveness of granulocytes to anaphylatoxins, C5a and C3a, in Churg-Strauss syndrome.

A 50-year-old man with Churg-Strauss syndrome showed granulocytes (GNLs) which generated more superoxide anion (O2-) than GNLs from healthy subjects following in vitro stimulation with C5a or C3a. Production of O2- subsided as the clinical symptoms improved with steroid treatment. A hyperresponsiveness of GNLs may be involved in this disorder.

Concentration of clarithromycin and 14-R-hydroxy-clarithromycin in plasma of patients with Mycobacterium avium complex infection, before and after the addition of rifampicin.

Clarithromycin (CAM) and rifampicin (RFP) have both been recognized to be effective antibiotic agents against Mycobacterium avium complex (MAC) infection. Rifamycin derivatives including RFP and rifabutin modulate the CAM metabolism by inducing the hepatic cytochrome p-450 3A4. To clarify the effect of RFP on the CAM metabolism, we measured the plasma concentration of CAM and 14-R-hydroxyclarithromycin (M-5), the major metabolite of CAM, in 9 patients suffering from MAC infection before and after the addition of RFP. After the addition of RFP, the mean plasma concentration of CAM significantly decreased, while that of M-5 did not. In addition, the amount of CAM + M-5 concentration also significantly decreased. As M-5 is less effective against MAC infection than CAM, more attention should thus be paid to the plasma CAM concentration in patients administered CAM and RFP concomitantly.

[Clinical studies on cefoxitin in the treatment of respiratory tract infections].

The clinical effects of cefoxitin (CFX) were studied in 31 cases of respiratory tract infections. The results were as follows: As for the clinical effects, CFX was excellent in 5 cases, good in 13, fair in 8 and poor in 5 out of 31 patients; the efficacy rate was 58.1%. The efficacy rate was 57.1% in bronchopneumonia, 61.1% in pneumonia and 50.0% in acute exacerbation of chronic respiratory tract infections. The efficacy rate was 70.6% in the group of 4 g/day or less and 42.9% in the group of 6 g/day or more. The efficacy rate was 50.0% in 6 cases that had not been responded to other antibiotics previously. As for side effects, skin eruption was observed in only 1 patient. No abnormality was observed in laboratory tests due to CFX. In conclusion, CFX is a useful drug in the treatment of respiratory tract infections.

[Clinical efficacy of imipenem/cilastatin sodium for respiratory infections in patients with lung cancer].

Imipenem/cilastatin sodium (IPM/CS) was administered to 102 patients with respiratory tract infections and lung cancer. Patients with other serious diseases were excluded and a total of 73 patients were enrolled. They were divided into 12 patients who underwent surgery (operated group) and 61 who did not (non-operated group); the latter group included 28 patients treated with anticancer agents or radiation therapy (treated group) and 33 untreated patients (untreated group). IPM/CS was effective in 75% of the patients, both with and without surgery. The drug was effective in 81% of the treated group, although many of the patients had Stage III or more advanced cancer, as well as bronchial occlusion. IPM/CS was also effective in 69% of the untreated group, although many of the patients have serious infections and a PS (Performance Status) of 3 or greater. Thus, IPM/CS treatment achieved good results. Bacteriological studies showed that 3 out of 4 strains in the operated group and 16 out of 18 in the non-operated group were eliminated. Safety was evaluated in all patients. Two patients (2%) experienced side effects and two others (2%) showed abnormal clinical findings, but the symptoms were mild and resolved after discontinuation or completion of therapy. In conclusion, IPM/CS was very effective for treating respiratory infections in patients with lung cancer.

[Pulmonary Mycobacterium Kansasii infection in the southern area of Fukuoka prefecture].

Cases of pulmonary infection caused by Mycobacterium kansasii (Mk) in our hospital located at the mid-northern area of the Kyushu district, which is in the southern part of Fukuoka prefecture were evaluated. Mk infection is not so rare in other areas of Japan, such as Tokyo and Kinki district, however, there has been no published report on the disease from the Kyusyu district. Therefore, the frequency and the clinical features of our cases of Mk infection were analyzed. During 17 years from 1982 to 1998, there were 14 patients of Mk infection out of 241 nontuberculous mycobacteriosis (NTM). There were 595 patients of culture-positive pulmonary tuberculosis without prior treatment (Tbc). The proportion of Mk/Tbc was 2.4% and that of Mk/NTM was 5.8%. During the period A (from 1982 to 1994) the ratio of Mk/Tbc was 5/462 (1.1%), while on the other side that of Mk/Tbc during the period B (from 1995 to 1998), it was 9/133 (6.8%), which was significantly (P < 0.01) higher compared with that in the period A. Although the patients of Mk infection in our hospital had been rare until 1994, from the results mentioned above, it was considered that the frequency of Mk infection in our hospital has increased to some extent since 1995. One of the characteristics in our cases was that the ratio of female (42.9%) was relatively high. All the female patients were considered to be compromised hosts. The results of the drug resistance tests were consistent with the other reports in our country. By the combination treatment including rifampicin as the major drug, the negative conversion of culture were obtained within 2 months in all our cases.

[Left Sylvian fissure meningioma in a one-year-eight-month old child].

We report a case of a Sylvian fissure meningioma in a one-year-eight-month old child who experienced the onset of a convulsive seizure. He had no neurological deficit and no developmental disorders. Computed tomography (CT) and magnetic resonance imaging (MRI) showed a large left temporal tumor which was well enhanced and without dural attachment. Angiography revealed a slight tumor stain in the left Sylvian fissure supplied by branches of the internal carotid artery. Total removal of the tumor was performed, and we found that the tumor had no dural attachment, but was strongly attached to the M2 segment of the left middle cerebral artery. Pathological examinations revealed it to be a fibrous meningioma without malignancy. This is the youngest case among the reported five pediatric deep Sylvian meningiomas. Introducing this case, we discuss the clinical features of pediatric meningiomas.

[Selective peripheral denervation for spasmodic torticollis involving the levator scapulae muscle].

Patients with laterocollis or rotatory type torticollis tend to show abnormal contraction of the levator scapulae muscle and the scalene muscles. These muscles are innervated from the anterior branches of the cervical spinal nerves. Because of this, the traditional Bertrand operation dealing with posterior branches does not adequately affect the symptoms of laterocollis. The authors report selective denervation of the levator scapulae muscle in three patients and discuss its rationale. All the three patients underwent denervation of both the C1-C6 posterior spinal rami and the branches from the C3 and C4 anterior rami to the levator scapulae muscle. We added myotomy of the scalene muscle in one patient, and denervation of the omohyoid muscle which is innervated from the ansa cervicalis and the descending branch of the hypoglossal nerve. The pre/post-operative Tsui scores were 12/4, 15/1, and 14/3 respectively. There were no complications. We conclude that selective peripheral denervation of the levator scapulae muscle is safe and effective in the treatment of laterocollic type torticollis.

[Purification and characterization of cerebroside sulfatase activator].

Cerebroside sulfatase (CSase) activator was isolated from human liver by acetone precipitation, anion-exchange chromatography, gel filtration and polyacrylamide gel electrophoresis. The CSase activator was a heat-stable protein with an isoelectric point of 4.54. Molecular weight (Mr) of the activator was estimated as 22,000 with the gel permeation and about 8,000 by gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native activator is a trimer of a subunit with Mr 8,000. The CSase activator formed a complex with an equimolar amount of cerebroside sulfate (CS), when examined by gel permeation experiments. The activator also bound to galactosylceramide and GM2 ganglioside but scarcely to GM1 ganglioside, and activated to some extent beta-N-acetyl-hexosaminidase A and beta-galactosidase, although the CSase activator could be clearly distinguished from the GM1 beta-galactosidase activator so far known. Though the affinity chromatography using glycolipid ligands, the CSase activator did not recognize sulfate group of CS, but appeared to have a relatively broad specificity for lipid-linked hexose.

[Experimental and clinical studies on ribosephosphate isomerase (author's transl)].

In order to know the clinical significance of serum ribose-5-phosphate isomerase (RPI), the activity of this enzyme was determined in sera of normal subjects and patients with hepatic disorders or malignant tumors. Experimentally, the enzyme activity in sera and liver tissue was followed in rats with acute hepatic damage induced by carbon tetrachloride (CCl4) or rats with hepatoma induced by 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). The following results were obtained: 1) Serum RPI activity increased markedly in rats with CCl4-induced liver damage, whereas the activity in liver tissue decreased, both being related with reciprocally. 2) In the early phase of acute hepatitis, serum RPI activity increased and gradually decreased thereafter. No significant increase was observed in other hepatic disorders. 3) Both serum and liver RPI activity increased in rats with hepatoma induced by 3'-Me-DAB. 4) An increase in serum RPI activity was seen in higher percentage in cancer patients. Higher enzyme activity and its higher incidence were observed in patients with hepatic metastasis or primary hepatoma than in patients without metastasis. From these results it is concluded that serum RPI activity as a diagnostic aid is useful in estimating clinical course of hepatic disorders and also in diagnosing malignant tumors, especially in substantiating a diagnosis of metastasis to the liver.