A case of systemic lupus erythematosus presenting with rectal ulcers as the initial clinical manifestation of disease.

Gastrointestinal involvement is often seen in patients with systemic lupus erythematosus (SLE). All parts of the gastrointestinal tract may be affected. However, rectal involvement at onset is rare. We describe here a case of SLE in which rectal ulcers due to vasculitis occurred as the initial manifestation of the disease without involvement of any other organ. The ulcers worsened, along with the appearance of lupus nephritis 5 years later When steroid therapy was initiated, there was rapid clinical and radiographic improvement. Our case suggests that rectal ulcer is a rare but important complication of SLE and can represent the initial and sole clinical manifestation of the disease.

Acute effect of human cardiotrophin-1 on hemodynamic parameters in spontaneously hypertensive rats and Wistar Kyoto rats.

There is considerable evidence to indicate that humoral factors play an important role in the development of left ventricular hypertrophy. Cardiotrophin-1 (CT-1) is a cytokine that has been shown to induce cardiac hypertrophy in a dose-dependent manner. The aim of the present study was to investigate the acute effect of CT-1 on hemodynamic parameters in spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) and to study the relationship between the plasma concentration of CT-1 and its hemodynamic effect. Ten-week-old SHR and age-matched WKY were used. Blood pressure (BP), heart rate (HR) and plasma concentration of CT-1 were measured both before and for 60 min after intravenous bolus injection of human CT-1 (10 microg/kg). CT-1 injection significantly decreased BP and significantly increased HR in SHR and WKY. There were significant differences in BP and HR between the two groups at all time points after injection. The lowest BP, highest HR and maximal plasma concentrations of CT-1 were observed in both groups within 10 min after injection. However, after converting the values into the percentage change from their respective baselines, there were no significant differences between the two groups in BP or HR at any time point. There was also no significant difference between the two groups at any time point in the plasma concentration of CT-1. This study indicates that CT-1 decreases BP and increases HR in both SHR and WKY. The most obvious change occurred within 10 min after injection. However, there was no significant difference in the hypotensive effect of CT-1 on 10-week-old SHR and WKY.

Elevated serum soluble CD44 level in sarcoidosis.

Sarcoidosis is a chronic multi-organ granulomatous disease of unknown etiology. Several studies have suggested an involvement of immunologic background in sarcoidosis. The lymphocyte surface marker CD44 is a multifunctional molecule which mediates the adhesion of lymphocytes to the extracellular matrix. Recently, we developed a system to quantitate soluble CD44 (sCD44) which we employed to determine serum and bronchoalveolar lavage fluid (BALF) levels of sCD44 to obtain further insights into immunologic aspects of sarcoidosis. Serum sCD44 levels were measured in 13 consecutive patients with sarcoidosis and 56 normal healthy controls using enzyme-linked immunoabsorbent assay. BALF sCD44 levels were also measured in 11 patients with sarcoidosis and 10 normal healthy controls. In patients with sarcoidosis, the serum sCD44 level was significantly higher than that of normal controls (348.5+/-164.2 ng/ml vs 145.4+/-22.9 ng/ml; p<0.001). Also BALF sCD44 levels tended to be higher in sarcoidosis than in normal controls (23.7+/-13.4 ng/ml vs 18.1+/-8.4 ng/ml), but no statistically significant difference was recognized. We also found that there was a positive correlation between the serum sCD44 and angiotensin converting enzyme (r=0.78). Our data indicate that sCD44 may be related to immunologic background and may be a useful new marker of sarcoidosis.

Human tissue distribution of TA02, which is homologous with a new type of aspartic proteinase, napsin A.

The N-terminal amino acid sequence of TA02 (molecular weight 35.0 kDa, isoelectric point 5.29), which is associated with primary lung adenocarcinoma, was determined and a fragment peptide was used to generate mouse monoclonal antibodies (mAbs) against TA02. The amino acid sequence suggested that TA02 might be homologous with napsin A, a new type of aspartic proteinase. In this context, we confirmed the expression of napsin A in primary lung adenocarcinoma using reverse-transcription polymerare chain reaction (RT-PCR) and showed that the TA02 mAbs reacted with glutathione-S-transferase (GST)-napsin A fusion protein. We concluded that TA02 is the same molecule as napsin A, and showed immunohistochemically that it is distributed mainly in type II pneumocytes, alveolar macrophages, renal tubules and exocrine glands and ducts in the pancreas. In particular, type II pneumocytes and alveolar macrophages showed high expression of TA02 among human normal tissues. In primary lung adenocarcinoma, 47 out of 58 (81.0%) primary lesions were positive. All well-differentiated adenocarcinomas except those of goblet cell type showed high expression of TA02. In addition, two out of seven (28.6%) large cell carcinomas showed low expression of TA02. The other histopathological types of primary lung cancer did not express TA02 at all. A few cases of renal cell cancer, pancreatic cancer, breast cancer, thyroid cancer, colon cancer and ovarian cancer showed low expression, but the staining patterns were completely different from that of primary lung adenocarcinoma, which showed a granular staining pattern. Our novel mAbs should be valuable for immunochemical detection of TA02/napsin A.

Calpain inhibitor inhibits secretory granule maturation and secretion of GH.

Clathrin- and AP-1-coated buds are present on immature secretory granules of endocrine cells that mature into clathrin-uncoated granules. The mechanism of clathrin and adaptor protein uncoating has remained obscure. Benzyloxycarbonyl-L-leucyl-L-leucinal (ZLLal), a calpain inhibitor, reduced growth hormone (GH) secretion with intracellular accumulation, in a GH-secreting rat pituitary tumor cell. Pulse and chase demonstrated that ZLLal retarded the turnover of clathrin (Clt.H) and adaptins. ZLLal-treatment co-immunoprecipitated the increased amounts of GH with Clt.H and adaptins compared to control cells, suggesting the intracellular accumulation of immature secretory granules. Clt.H and adaptins were limited-proteolyzed by m-calpain in vitro, indicating that calpain may be involved partly in the maturation of secretory granules in endocrine cells via the process of clathrin uncoating.

Linkage of mutant alleles of CYP2C18 and CYP2C19 in a Japanese population.

The coincidence of mutated alleles of CYP2C18 and CYP2C19 was studied in 154 Japanese subjects. The mutant alleles of CYP2C18 studied were CYP2C18m1 (T204 --> A substitution in exon 2) and CYP2C18mFR (A-460 --> T substitution in the 5'-flanking region), and those of CYP2C19 were CYP2C19m1 (G689 --> A substitution in exon 5) and CYP2C19m2 (G636 --> A substitution in exon 4). They were identified by polymerase chain reaction and restriction fragment length polymorphism. The results indicate that genotypes of CYP2C18m1 and CYP2C18mFR are completely coincident with those of CYP2C19m2 and CYP2C19m1, respectively. The finding suggests that the mutations of CYP2C18 and CYP2C19 examined in the present study are very closely linked with each other (i.e. CYP2C18m1 vs CYP2C19m2 and CYP2C18mFR vs CYP2C19m1), at least in a Japanese population.

Serum soluble CD44 levels for monitoring disease states in acute leukemia and myelodysplastic syndromes.

To determine the clinical implications of soluble CD44 (sCD44) levels in hematologic neoplasias, we developed an enzyme-linked immunosorbent assay for sCD44 using two monoclonal antibodies to the standard 90 kDa form, and assessed the serum concentration of sCD44 in normal healthy volunteers, patients with acute leukemia, myelodysplastic syndromes (MDS), and those with chronic myeloid leukemia (CML). Compared to that in normal individuals (n=51; 145. 1 24.6 ng/ml), the serum sCD44 level was significantly elevated in patients with acute myeloid leukemia (AML; n=18; 331.9 99.0 ng/ml, P=0.0001), acute lymphoid leukemia (ALL; n=16; 551.3 427.8 ng/ml, P=0.0001) and CML (n=18; 262.0 97.5 ng/ml, P=0.0001). The sCD44 level was slightly elevated in patients with MDS (n=43; 173.8 54.9 ng/ml, P=0.0071). In patients with acute leukemia, serum sCD44 concentrations decreased significantly in response to treatment and reached nearly normal levels after complete remission (P=0.0005 in AML and P=0.0032 in ALL). The sCD44 levels in patients with MDS increased after they developed acute leukemia, whereas no significant difference in sCD44 levels was observed between the chronic and the blastic phases in patients with CML. Our results indicate that serum sCD44 levels may be a useful marker for monitoring response to treatment and disease progression, especially in acute leukemia.

Serum concentrations of free ubiquitin and multiubiquitin chains.

Ubiquitin, which can conjugate with cellular proteins, is classified into two forms: free ubiquitin and multiubiquitin chains. The latter is active as a signal for degradation of the targeted proteins. We found both forms in human serum and, using two immunoassays, quantitated them in sera from healthy subjects and patients with some diseases. Because of putative leakage of erythrocyte ubiquitin, hemolytic serum and serum obtained after long incubation (> 1-2 h) of blood at room temperature were excluded. Serum concentrations of multiubiquitin chains and free ubiquitin were substantially higher in rheumatoid arthritis and hemodialysis patients, respectively, than healthy subjects. Additionally, in acute viral hepatitis, serum multiubiquitin chain concentrations were increased in the acute phase, decreased in the recovery phase, and correlated with alanine and aspartate aminotransferase activities (r = 0.676 and 0.610, P < 0.0001 and < 0.001, respectively). Therefore, serum ubiquitin may have prognostic value.

[Laparoscopically assisted surgery for Crohn's disease].

PURPOSE: Surgical treatment is often required by patients with Crohn's disease who have intestinal stricture or fistula. Minimally invasive laparoscopic surgery appears to be useful in such patients. Eight cases of intestinal stricture and/or fistula treated by laparoscopic surgery are reported. METHODS: In 1997, laparoscopic surgery was attempted and successfully completed in 18 patients Crohn's disease presenting with intestinal stricture and/or fistula, following strict nutritional therapy. Of the 8 patients with a fistula, one had an gastrocdic fistula, one had an ileovesical fistula, two had an colic fistula and three had ileoileal fistulas. Ileovesical, ileorectal and gastrocolic fistulas were divided with an intracorponeal automatic stapling device. Fourteen patients underwent ileocecal resection and four underwent ileal resection with laparoscopic assistance. RESULTS: Postoperative pain was mild, oral intake was started an average of 1.4 days after operation. The patients were discharged an average of 8 days after operation. No complication was identified. CONCLUSION: If Crohn's disease is treated adequately with conservative therapy, laparoscopic surgery is relatively easy, even when there are adhesions and fistulas. Since the incision is extremely small with this method, postoperative adhesions are minimized. This procedure has the advantage of allowing repeated laparoscopic surgery with minimal morbidity. Minimal invasiveness and rapid return to normal activity may make laproscopic surgery the treatment of choice for patients with Crohn's disease who require surgical treatment.

Ability of ubiquitin radioimmunoassay to discriminate between monoubiquitin and multi-ubiquitin chains.

Free ubiquitin (mainly monoubiquitin) and multi-ubiquitin chains coexist in eukaryote cells and serve distinct cellular roles. However, any immunoassay systems established previously have not been proved to be applicable for measuring the former without cross-reactive responses with the latter. For this purpose, we developed a radioimmunoassay specific to monoubiquitin by employing antiserum US-1 against ubiquitin. In this assay, ubiquitin-protein conjugates, prepared by a reticulocyte lysate fraction II and fractionated on Moro Q and Superdex 200 columns, exhibited practically no cross-reactivity. The cross-reactivity of fractionated ubiquitin-lysozyme conjugates was also analyzed as a function of their multi-ubiquitin chain size. As a result, the larger the conjugates were found to be, the weaker were the cross-reactive responses they showed, and the multi-ubiquitin chains (n > approx. 20) were substantially unreactive in the radioimmunoassay. By using the radioimmunoassay, heat-shock-induced decrease in the level of cellular free (mono)ubiquitin was detected. In addition, the standard preparation of multi-ubiquitin chains was not cross-reactive in all other five radioimmunoassays employing distinct antibodies to ubiquitin (four antisera and a monoclonal antibody). These data suggest that radioimmunoassays employing ubiquitin antibodies raised by the general methods can discriminate between monoubiquitin and multi-ubiquitin chains and quantitate cellular free ubiquitin.

Regulation of pyridoxal-5'-phosphate level by biogenic amines in mouse brain.

We investigated the relationship between the concentration of pyridoxal-5'-phosphate (PLP) and biogenic amine in mouse brain. The production of PLP from pyridoxal (PL) by pyridoxal kinase (PLK) was inhibited by the addition of dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT), but not by that of epinephrine and N-acetyl-serotonin. DA and NE were combined with PLP by a non-enzymatic reaction, whereas 5-HT was bound only slightly with PLP. The conjugated product of PLP with DA was also detected by HPLC analysis when PLK activity was assayed using PL as a substrate in the presence of DA. In an in vivo investigation, the depletion of DA and 5-HT in mouse brain after an intraperitoneal injection of 5 mg/kg reserpine, led to slight elevation of the PLP level to 120% of the control level. By contrast, the increase in DA in the brain caused by intraperitoneal administration of 150 mg/kg L-DOPA caused the PLP concentration to decrease to 70% of the control level. However, no change in PLK activity in the brain was observed when the mice were treated with either reserpine or L-DOPA. These results suggested that the level of PLP in mouse brain was partly regulated by the concentration of biogenic amines, such as DA, NE and 5-HT, without apparent induction of PLK.

Work and power outputs determined from pedalling and flywheel friction forces during brief maximal exertion on a cycle ergometer.

Work and power outputs during short-term, maximal exertion on a friction loaded cycle ergometer are usually calculated from the friction force applied to the flywheel. The inertia of the flywheel is sometimes taken into consideration, but the effects of internal resistances and other factors have been ignored. The purpose of this study was to estimate their effects by comparing work or power output determined from the force exerted on the pedals (pedalling force) with work or power output determined from the friction force and the moment of inertia of the rotational parts. A group of 22 male college students accelerated a cycle ergometer as rapidly as possible for 3 s. The total work output determined from the pedalling force (TWp) was significantly greater than that calculated from the friction force and the moment of inertia (TWf). Power output determined from the pedalling force during each pedal stroke (SPp) was also significantly greater than that calculated from the friction force and the moment of inertia. Percentage difference (% diff), defined by % diff = ¿(TWp - TWf)/TWf¿ x 100, ranged from 16.8% to 49.3% with a mean value of 30.8 (SD 9.1)%. It was observed that % diff values were higher in subjects with greater TWp or greater maximal SPp. These results would indicate that internal resistances and other factors, such as the deformation of the chain and the vibrations of the entire system, may have significant effects on the measurements of work and power outputs. The effects appear to depend on the magnitudes of pedalling force and pedal velocity.

Immunoassay for the quantification of intracellular multi-ubiquitin chains.

A sandwich ELISA has been developed to measure intracellular levels of multi-ubiquitin chains. The mixture of multi-ubiquitin chains, prepared in vitro by incubation of ubiquitin (plus 125I-ubiquitin) and lysozyme with ubiquitin-ligating enzymes and ATP, was partially purified and established as a standard named the multi-ubiquitin-chain reference preparation 1 (MUCRP1). The concentration of MUCRP1 was calculated from the recovered radioactivity of 125I-ubiquitin. All measurements by the ELISA were expressed in terms of MUCRP1. The ELISA showed good sensitivity (98 pg/ml), precision (intra-assays < 6%) and reproducibility (interassay < 9%). In addition, there was no substantial cross-reaction with mono-, di- and tri-ubiquitin, or mono-ubiquitinated and di-ubiquitinated lysozyme in the ELISA, and large multi-ubiquitin chains (n > approximately 6) may be fully reactive. These results combined with excellent results in the recovery and dilution tests guarantee accurate measurement of multi-ubiquitin chains in cell extracts prepared with a lysis buffer (water soluble) or the buffer supplemented 8 M urea (urea soluble). The level of the water-soluble multi-ubiquitin chains in reticulocytes was lower than that of erythrocytes, but the urea-soluble chain level was higher in the reticulocytes. Heat-shock treatment of HeLa cells increased the urea-soluble multi-ubiquitin chains. These data indicate that this ELISA provides a useful and reliable approach to the study of intracellular multi-ubiquitin-conjugate turnover.

Gene expression in tobacco low-nicotine mutants.

Two nuclear genes, Nic1 and Nic2, regulate nicotine levels in tobacco. nic1 and nic2 are semidominant mutations in Burley 21 that reduce leaf nicotine levels and the activities of multiple enzymes in the nicotine pathway and simultaneously increase polyamine levels in cultured roots. Cultured roots homozygous for both mutations were used to isolate two cDNAs by subtraction hybridization; the transcript levels of these two cDNAs were much lower in the mutant roots than in the wild-type roots. The A411 gene encodes a 41-kD protein with considerable homology to mammalian spermidine synthase, whereas the A622 gene encodes a 35-kD protein with high homology to isoflavone reductase. When these genes were expressed in Escherichia coli, A411 had no spermidine synthase activity but did show putrescine N-methyltransferase activity, which is the first enzyme committed to the nicotine biosynthetic pathway, and A622 did not show isoflavone reductase activity. Both the methyltransferase and A622 genes are predominantly expressed in the root, and their expression levels in cultured roots are coordinately decreased by the nic mutations in the order of wild type > nic2 > nic1 > nic1 nic2. Removal of tobacco flower heads and young leaves rapidly and coordinately induced both genes in the root. Further, exogenous supply of auxin down-regulated both genes in cultured tobacco roots. These results suggest that Nic1 and Nic2 are regulatory genes for nicotine biosynthesis.

Putrescine N-Methyltransferase in Cultured Roots of Hyoscyamus albus: n-Butylamine as a Potent Inhibitor of the Transferase both in Vitro and in Vivo.

Biosynthesis of tropane alkaloids is thought to proceed by way of the diamine putrescine, followed by its methylation by putrescine N-methyltransferase (PMT; EC 2.1.1.53). High PMT activities were found in branch roots and/or cultured roots of several solanaceous plants. PMT was partially purified and characterized from cultured roots of Hyoscyamus albus that contain hyoscyamine as the main alkaloid. Initial velocity studies and product inhibition patterns of PMT are consistent with an ordered bi-bi mechanism, in which the K(m) values for putrescine and S-adenosyl-l-methionine are 277 and 203 mum, respectively, and the K(i) value for S-adenosyl-l-homocysteine is 110 mum. PMT efficiently N-methylated amines that have at least two amino groups separated by three or four methylene groups. Monoamines were good competitive inhibitors of PMT, among which n-butylamine, cyclohexylamine, and exo-2-aminonorbornane were most inhibitory, with respective K(i) values of 11.0, 9.1, and 10.0 mum. When n-butylamine was fed to root cultures of H. albus, the alkamine intermediates (tropinone, tropine, and pseudotropine) drastically decreased at 1 mm of the exogenous monoamine, and the hyoscyamine content decreased by 52% at 6 mm, whereas the contents of 6beta-hydroxyhyoscyamine and scopolamine did not change. Free and conjugated forms of polyamines were also measured. The n-butylamine treatment caused a large increase in the putrescine content (especially in the conjugated pool), and the spermine content also increased slightly, whereas the spermidine content decreased slightly. The increase in the putrescine pool size (approximately 40 nmol/mg dry weight) was large enough to account for the decrease in the total alkaloid pool size. Similar results were also obtained in root cultures of Datura stramonium. These studies further support the role of PMT as the first committed enzyme specific to alkaloid biosynthesis.

[Elevation of serum fucosyltransferase activities in malignant diseases--a sensitive tumor marker?].

Fucosyltransferase (FT) is considered to be one of the most important glycosyltransferases responsible for the synthesis of cancer-associated carbohydrate chains such as CA19-9 and SLX. To determine whether FT is a sensitive tumor marker, we measured the enzyme activity of FT in sera from 136 cancer patients, 14 patients with benign diseases and 59 healthy controls, by using PA (pyridylamino)-labeled type II biantennary oligosaccharide derived from human serotransferrin as an acceptor substrate. Serum FT activity was significantly elevated in patients with cancer compared to healthy controls. Analysis of the enzyme products using HPLC and various fucosidases with different specificity revealed that alpha 1----3 FT was responsible for most of the elevation of the enzyme activity in sera from cancer patients. It should be stressed that the alpha 1----3FT derived from cancer patients transferred fucose to terminal lactosamine residues of type II biantennary oligosaccharides already attached to sialic acid. This indicates that the substrate specificity is clearly different from that reported in normal sera and tissues. In addition to alpha 1----3FT, some glycosidases including fucosidase were also elevated in sera from cancer patients.

Beneficial effect of nipradilol (K-351) on acute myocardial ischemia. Study of the relationship between regional myocardial blood flow and energy metabolism.

To examine the effects of nipradilol on ischemic myocardium, experiments were performed on regional myocardial blood flow (MBF) and energy metabolism in anesthetized, open-chest dogs. Nipradilol at a dose of 0.3 mg/kg was i.v.-administered 10 min after coronary ligation. MBFs at various sites, including ischemic and non-ischemic areas, were determined by the hydrogen gas clearance method. The levels of ATP and creatine phosphate (CP) at the site of MBF determination were measured 60 min after ligation, and mitochondrial function (RCI, QO2) in the ischemic and non-ischemic areas was determined. Following nipradilol administration, aortic pressure and heart rate were significantly lowered. In ischemic areas with MBF below 40 ml/min/100 g, nipradilol had no influence on MBF. However, the tissue level of ATP in nipradilol treated hearts was significantly higher as compared with untreated hearts. In the area of mild ischemia with MBF of 40-60 ml/min/100 g, nipradilol preserved the tissue ATP and CP levels in spite of a decrease in MBF. Moreover, an inhibition of the decrease in mitochondrial respiratory function was observed in ischemic areas with MBF below 20 ml/min/100 g. Thus, nipradilol administered following ischemia preserved ATP content and mitochondrial function in the ischemic myocardium with reduction of heart rate and aortic pressure. This suggests that nipradilol exerts a cardioprotective effect in acute ischemia. It seems that the cardioprotective effect is due to a decrease in myocardial oxygen demand and preservation of mitochondrial function.