Isoquinoline alkaloids from cell suspension cultures of Fumaria capreolata

Fumaria capreolata was taken into cell suspension culture. The culture yielded a biomass of about 12 g dry weight per liter of medium; the dried cells contained ca. 0.1% of alkaloids. Besides choline, the following ten known isoquinoline alkaloids were isolated from the cell extract in crystalline form: coptisine, dehydrocheilanthifoline; (+)-isoboldine; magnoflorine; N-methylcoclaurine; (+)-reticuline; (–)-pallidine; protopine; sanguinarine; (–)-scoulerine. This is the most diverse isoquinoline alkaloid spectrum thus far published for a cell suspension culture.     

Purification and properties of glucose-6-phosphate dehydrogenase from Bacillus subtilis spores.

Glucose-6-phosphate dehydrogenase [D-glucose-6-phosphate: NADP oxidoreductase, EC. 1. 1. 1. 49] obtained from spores of Bacillus subtilis PCI 219 strain was partially purified by filtration on Sephadex G-200, ammonium sulfate fractionation and chromatography on DEAE-Sephadex A-25 (about 54-fold). The optimum pH for stability of this enzyme was about 6.3 and the optimum pH for the reaction about 8.3. The apparent Km values of the enzyme were 5.7 X 10(-4) M for glucose-6-phosphate and 2.4 X 10(-4) M for nicotinamide adenine dinucleotide phosphate (NADP). The isoelectric point was about pH 3.9. The enzyme activity was unaffected by the addition of Mg++ or Ca++. The inactive glucose-6-phosphate dehydrogenase obtained from the spores heated at 85 C for 30 min was not reactivated by the addition of ethylenediaminetetraacetic acid, dipicolinic acid or some salts unlike inactive glucose dehydrogenase.     

Synthesis of 2-deoxy-4-O-phosphono-3-O-tetradecanoyl-2-[(3R)- and (3S)-3-tetradecanoyloxytetradecanamido]-D-glucose: a diastereoisomeric pair of 4-O-phosphono-D-glucosamine derivatives (GLA-27) related to bacterial lipid A

The diastereoisomeric, 4-O-phosphono-D-glucosamine derivatives named in the title have been synthesized, starting from benzyl 2-amino-2-deoxy-4,6-O-isopropylidene-beta-D-glucopyranoside and (3RS)-3-hydroxytetradecanoic acid.     

Effects of hemolysis, hematocrit, RBC swelling, and flow rate on light scattering by blood in a 0.26 cm ID transparent tube

The intensity of light scattering by blood in a tube of diameter 0.26 cm was measured with an apparatus devised by us at different angles on an incident cross-sectional plane. Changes in angular distribution of light intensity associated with hemolysis, and changes in hematocrit (Ht), red blood cell (RBC) swelling, and flow rate were plotted on polar coordinates. The dyssymmetry index, defined as the ratio of the intensity of light at 45 degrees to that at 135 degrees, was used to characterize the shape of the diagrams of light scattering. The index decreased with Ht, flow rate, but increased with RBC swelling. It is concluded that light scattering by blood requires intactness of the RBC membrane. Even when the cell membrane is intact, light scattering is subject to changes with the flow rate of blood, presumably due to RBC aggregation.     

Intravesical treatment of bladder cancer with recombinant human interferon-β

Summary In order to examine its clinical efficacy, recombinant human interferon- (rIFN-) was instilled intravesically into 51 patients with superficial bladder cancer. Ten patients, who received intermittent intravesical instillation at a dose of (3–36) × 10⁶ U rIFN- on days 1–3 every week, showed no response. Thirty-two patients received intravesical instillation at a dose of (3–36) × 10⁶ U every day for 10–20 days. Eight patients showed partial response, indicating an efficacy rate of 25%. Nine patients received divided doses of 18 × 10⁶ U twice a day every day for 10–20 days. Six patients showed partial response, indicating an efficacy rate of 67%. This value was significantly higher than that obtained by administering divided doses. The response to intravesical instillation therapy with rIFN- varies with treatment protocol. Frequent and longer exposure to rIFN- may induce better regression of superficial bladder cancer. Six incidences of side-effects were found in five cases (9.8%): pollakiuria in one, pain on micturition in two, fever in two, and eruption in one case. All of these side-effects were slight and reversible after drug withdrawal. Laboratory tests showed only a few changes with low severity. Thus, rIFN- is potentially a new drug for instillation therapy of superficial bladder cancer, in view of the absence of adverse effects.     

Anti-pituitary antibodies in patients with hypopituitarism and their families: longitudinal observation.

In an attempt to investigate the clinical significance of anti-pituitary antibodies in patients with hypopituitarism, anti-pituitary antibody in plasma was examined in 10 such patients (7 cases of isolated ACTH deficiency, 1 of partial hypopituitarism, and 2 of Sheehan's syndrome), on two or three occasions with an interval of more than 6 months (longitudinal study). In a total of 16 relatives of these 4 patients (2 cases of Sheehan's syndrome, one in each of partial hypopituitarism and isolated ACTH deficiency) and one patient not involved in the longitudinal study, anti-pituitary antibodies were also examined (family study). Anti-pituitary antibodies reacting with rat pituitary cytoplasmic antigens (pituitary cell antibodies: PCA) and pituitary cell surface antibodies (PCSA) reacting with GH3 cells and/or AtT-20 cells were measured with indirect immunofluorescence. The longitudinal study revealed the disappearance of antibodies in 3 patients, 2 PCA positive and one both PCA and PCSA positive. In 3 patients, altered antibody titers or a newly appearing antibody were found during the follow-up period. In 4 patients, the pituitary antibodies were negative during the follow-up periods. Of 16 family members studied, positive PCA was found in 3 members (2 in the families of patients with PCA positive Sheehan's syndrome, and 1 in the family of the patients with PCA positive partial hypopituitarism). Positive PCSA was found in 4 members (one in each of families of patients with partial hypopituitarism and isolated ACTH deficiency and of two cases of Sheehan's syndrome), and weakly positive PCSA was found in one family member of a patients with PCA positive Sheehan's syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Demonstration that a human 26S proteolytic complex consists of a proteasome and multiple associated protein components and hydrolyzes ATP and ubiquitin-ligated proteins by closely linked mechanisms.

It is known that two types of high-molecular-mass protease complexes are present in the cytosol of mammalian cells; a 20S latent multicatalytic proteinase named the proteasome, and a large proteolytic complex with an apparent sedimentation coefficient of 26S that catalyzes ATP-dependent breakdown of proteins conjugated with ubiquitin. In this work, we first demonstrated that a low concentration of SDS was required for activation of the latent proteasome, whereas the 26S complex degraded substrates for proteasomes in the absence of SDS. Moreover, the 26S complex was greatly stabilized in the presence of 2 mM ATP and 20% glycerol. Based on these characteristics, we next devised a novel procedure for purification of the 26S proteolytic complexes from human kidney. In this procedure, the proteolytic complexes were precipitated from cytoplasmic extracts by ultracentrifugation for 5 h at 105000 x g, and the large 26S complexes were clearly separated from the 20S proteasomes by molecular-sieve chromatography on a Biogel A-1.5 m column. The 26S enzyme was then purified to apparent homogeneity by successive chromatographies on hydroxyapatite and Q Sepharose, then by glycerol density-gradient centrifugation. Electrophoretic and immunochemical analyses showed that the purified human 26S complex consisted of multiple subunits of proteasomes with molecular masses of 21-31 kDa and 13-15 protein components ranging in molecular mass over 35-110 kDa, which were directly associated with the proteasome. The purified 26S proteolytic complex degraded 125I-labeled lysozyme-ubiquitin conjugates in an ATP-dependent manner. The 26S enzyme also showed high ATPase activity, which was copurified with the complex. Vanadate and hemin strongly inhibited not only ATP cleavage, but also ATP-dependent breakdown of ubiquitinligated proteins, suggesting that the 26S complex hydrolyzes ATP and ubiquitinated proteins by closely linked mechanisms. These findings indicate that the 26S complex consists of a proteasome with proteolytic function and multiple other components including an ATPase that regulates energy-dependent, ubiquitin-mediated protein degradation.     

Monoclonal antibody against platelet thrombospondin decreases erythrocyte aggregation rate.

The effect of thrombospondin, a major glycoprotein in the platelet alpha-granule, on the erythrocyte aggregation rate was investigated. Venous blood was sampled from 8 healthy male volunteers and anticogulated with 1.1 mg/ml EDTA(K2). The erythrocyte aggregation rate of each blood sample was measured with a whole-blood erythrocyte aggregometer before and after incubation with murine monoclonal antibody against human platelet thrombospondin. After 15 min incubation, the erythrocyte aggregation rate exhibited a significant decrease to 0.055 +/- 0.022/s, representing 71.9 +/- 8.7% of the control value (0.075 +/- 0.028/s) (p less than 0.0005). The results obtained suggest that thrombospondin may participate in the control of erythrocyte aggregability in the circulating blood.     

Noisy Interlimb Coordination Can Be a Main Cause of Freezing of Gait in Patients with Little to No Parkinsonism

Freezing of gait in patients with Parkinson’s disease is associated with several factors, including interlimb incoordination and impaired gait cycle regulation. Gait analysis in patients with Parkinson’s disease is confounded by parkinsonian symptoms such as rigidity. To understand the mechanisms underlying freezing of gait, we compared gait patterns during straight walking between 9 patients with freezing of gait but little to no parkinsonism (freezing patients) and 11 patients with Parkinson’s disease (non-freezing patients). Wireless sensors were used to detect foot contact and toe-off events, and the step phase of each foot contact was calculated by defining one stride cycle of the other leg as 360°. Phase-resetting analysis was performed, whereby the relation between the step phase of one leg and the subsequent phase change in the following step of the other leg was quantified using regression analysis. A small slope of the regression line indicates a forceful correction (phase reset) at every step of the deviation of step phase from the equilibrium phase, usually at around 180°. The slope of this relation was smaller in freezing patients than in non-freezing patients, but the slope exhibited larger step-to-step variability. This indicates that freezing patients executed a forceful but noisy correction of the deviation of step phase, whereas non-freezing patients made a gradual correction of the deviation. Moreover, freezing patients tended to show more variable step phase and stride time than non-freezing patients. Dynamics of a model of two coupled oscillators interacting through a phase resetting mechanism were examined, and indicated that the deterioration of phase reset by noise provoked variability in step phase and stride time. That is, interlimb coordination can affect regulation of the gait cycle. These results suggest that noisy interlimb coordination, which probably caused forceful corrections of step phase deviation, can be a cause of freezing of gait.     

An endogenous electrophile that modulates the regulatory mechanism of protein turnover: inhibitory effects of 15-deoxy-Delta 12,14-prostaglandin J2 on proteasome

Prostaglandin D(2) (PGD(2)), a major cyclooxygenase product in a variety of tissues and cells, readily undergoes dehydration to yield electrophilic PGs, such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). We have previously shown that 15d-PGJ(2) potently induces apoptosis of SH-SY5Y human neuroblastoma cells via accumulation of the tumor suppressor gene product p53. In the study presented here, we investigated the molecular mechanisms involved in the 15d-PGJ(2)-induced accumulation of p53. It was observed that 15d-PGJ(2) potently induced p53 protein expression but scarcely induced p53 gene expression. In addition, exposure of the cells to 15d-PGJ(2) resulted in an accumulation of ubiquitinated proteins and in a significant inhibition of proteasome activities, suggesting that 15d-PGJ(2) acted on the ubiquitin-proteasome pathway, a regulatory mechanism of p53 turnover. The effects of 15d-PGJ(2) on the protein turnover were attributed to its electrophilic feature, based on the observations that (i) the reduction of the double bond in the cyclopentenone ring of 15d-PGJ(2) virtually abolished the effects on protein turnover, (ii) overexpression of an endogenous redox regulator, thioredoxin 1, significantly retarded the inhibition of proteasome activities and accumulations of p53 and ubiquitinated proteins induced by 15d-PGJ(2), and (iii) treatment of SH-SY5Y cells with biotinylated 15d-PGJ(2) indeed resulted in the formation of a 15d-PGJ(2)-proteasome conjugate. These data suggest that the modulation of proteasome activity may be involved in the mechanism responsible for the accumulation of p53 and subsequent induction of apoptotic cell death induced by 15d-PGJ(2).