Putative mechanisms of action of probucol on high-density lipoprotein apolipoprotein A-I and its isoproteins kinetics in rabbits

Probucol is a widely prescribed lipid-lowering agent, the major effects of which are to lower cholesterol in both low- and high-density lipoproteins (LDL and HDL, respectively). The mechanism of action of probucol on HDL apolipoprotein (apo) A-I kinetics was investigated in rabbits, with or without cholesterol feeding. 125I-labeled HDL was injected intravenously, and blood samples were taken periodically for 6 days. Kinetic parameters were calculated from the apo A-I-specific radioactivity decay curves. Fractional catabolic rate (FCR) and synthetic rate (SR) of apo A-I in rabbits fed a normal chow and normal chow with 1% probucol were similar. Apo A-I FCR of the rabbits fed 0.5% cholesterol was significantly increased but there were no changes in SR, compared to findings in the normal chow-fed group. Apo A-I FCR of the rabbits fed 1% probucol with 0.5% cholesterol (both 1 month and 2 months) was significantly increased compared to findings in rabbits fed the normal chow as well as 0.5% cholesterol diet group, while SR of apo A-I was significantly reduced in the former groups. Kinetics at 1 month after discontinuation of 1% probucol (under cholesterol feeding) showed a similar FCR of HDL-apo A-I to that of the rabbits fed 0.5% cholesterol, but the SR of apo A-I remained lower. Apo A-I isoproteins kinetics assessed by autoradiography of isoelectric focusing slab gels showed that the synthesis of proapo A-I was significantly reduced in the 1% probucol with 0.5% cholesterol administered, compared to the 0.5% cholesterol group. Thus, the action of probucol on HDL apo A-I kinetics was only prominent in case of higher serum cholesterol levels. The decreased HDL or apo A-I seen with probucol was apparently the result of an increase in FCR and a decrease in SR of HDL-apo A-I. A decreased synthesis of apo A-I remained evident even 1 month after discontinuing probucol. The action of probucol on the intracellular synthetic processes of apo A-I was revealed by the reduced synthesis of proapo A-I.     

Development of a Sensitive Microbioassay Using Amplified Cultivation1

A novel ultramicro microbioassay was developed. The present method, referred to as amplified cultivation, is composed of two successive kinds of culture. The first culture is performed until a maximum difference is obtained in the ratio of numbers of viable cells appearing in a basal medium and that contained in a nutrient. The second culture is continued after adding a complete medium to the cultures and stopped just after the growth of microorganisms in the blank reaches a certain detectable limit, e.g., by optical density or acid production. Lactose was analyzed by this amplified cultivation method with an extremely low concentration of viable cells of Bifidobacterium bifidum N4 as the inoculum. The detectable limits of lactose were 0.05 μg and 1 μg by the test tube method and by the pulp disc method respectively, with an increase in sensitivity by a factor of more than 10³ compared with the corresponding conventional microbioassays. Moreover, a relationship between the diameters of the growth zones and the logarithm of the amount of lactose was achieved in a range of 50–1000 μg of lactose in the pulp disc method.     

Isolation and Characterization of the Serotype g Carbohydrate Moiety from an Enzyme Lysate of Streptococcus mutans 6715 Cell Walls

The serotype-specific carbohydrate moiety of Streptococcus mutans was isolated by mild degradation of purified cell walls with a cell-wall lytic enzyme. Cell walls of serotype g S. mutans strain 6715 were digested with M1 enzyme, an endo-N-acetylmuramidase purified from culture supernatants of Streptomyces globisporus strain 1829. The enzyme lysate of the cell walls was applied to a CM Sephadex C-25 column to remove the M1 enzyme from the cell wall lysate and then subjected to Sephadex G-100 column chromatography. Carbohydrate antigens with serotype g specificity, designated M1g, and a peptidoglycan—polysaccharide complex lacking serotype specificity (M1PG) were separated. Purified serotype g antigen was also obtained by autoclaving the S. mutans 6715 whole cells in saline at 120 C for 30 min. The extract was applied to a DEAE Sephadex A-25 column to remove nucleic acids and teichoic acids. The unbound peak fraction was concentrated and re-chromatographed on a Bio-Gel P-100 column. The void volume fraction contained serotype g carbohydrate and was designated RRg antigen. M1g and RRg antigens formed a band of identity with anti-serotype g serum by immunodiffusion. These antigens were composed mainly of galactose, glucose, and rhamnose at an approximate weight ratio of 8 : 4 : 1, while constituent sugars of M1PG consisted of rhamnose and glucose, with no detectable galactose. M1g also contained peptidoglycan residues other than threonine, an interpeptide bridge component of the native cell wall peptidoglycan. Marked inhibition of the quantitative precipitin reaction between M1g and anti-serotype g serum was obtained with melibiose and galactose, which suggests that the immunodeterminant of the serotype g carbohydrate is an α-linked galactose-glucose terminal linkage.     

Polyamine distribution and the potential to form novel polyamines in phytopathogenic agrobacteria

Abstract Polyamines were analyzed in 4 species of genus Agrobacterium . Not only putrescine, spermidine and spermine, but also homospermidine and thermospermine were found in A. tumefaciens, A. radiobacter, A. rubi and A. rhizogenes . Trace amounts of aminopropylhomospermidine were also observed. Norspermidine and norspermine were formed from diamonorpropane added to the medium. Aminopropylcadaverine and its aminopropyl derivative(s) (aminopentylnorspermidine and N,N '-bis(3-aminopropyl) cadaverine) were produced from the supplemented cadaverine. A strain of A. rhizogenes normally contains only putrescine and homospermidine; no other diamines, triamines and tetraamines were synthesized.     

Microaerophilic property of Actinobacillus actinomycetemcomitans in fructose-limited chemostat cultures

Abstract The effect of oxygen on the growth, metabolism, and leukotoxin production of Actinobacillus actinomycetemcomitans 301-b was examined using a chemostat equipped with a redox potential control system. Steady states were obtained with fructose-limited cultures grown at a dilution rate of 0.1 h⁻¹ under strictly anaerobic ( E _h=−460 mV) and microaerobic conditions ( E _h≤ 150 mV) but not under highly aerated conditions ( E _h≥ 100 mV). The optimum growth was recorded at E _h=−300 to − 200 mV and the recorded Y _fructose value was about 1.3 times the Y _fructose of anaerobic cultures. Although the organism contains a respiratory chain, the increased Y _fructose under the microaerobic conditions might result from the increased substrate-level phosphorylation at the site of acetate kinase but not from electron transport phosphorylation. After passing threshold aeration ( E _h=−100 mV), the culture yielded a variant with transparent colony morphology. Under anaerobic conditions, the Y _fructose of the variant was about 1.6 times that of the original opaque colony-forming cells. The optimum growth of the variant was also recorded at E _h=− 300 to − 200 mV. In both types of cells, the production of leukotoxin reached a maximum at E _h=−350 to − 200 mV. These findings suggested the microaerophilic nature of A. actinomycetemcomitans .     

Identification and determination of urinary acetylpolyamines in cancer patients by electrospray ionization and time-of-flight mass spectrometry

A method for the quantification of acetylpolyamines, N¹,N¹²-diacetylspermine (DiAcSpm), monoacetylspermidine (AcSpd), and N¹,N⁸-diacetylspermidine (DiAcSpd), identifying each compound simultaneously, was developed with the goal of evaluating these acetylpolyamines as potential biomarkers of cancer. The method consists of prepurification of acetylpolyamines in urine with commercially available cartridges and derivatization with heptafluorobutyric (HFB) anhydride. HFB derivatives of acetylpolyamines were determined simultaneously using ¹⁵N-labeled acetylpolyamines as internal standards by electrospray ionization and time-of-flight mass spectrometry (ESI-TOF MS). After the method was validated, the urinary acetylpolyamines of 38 cancer patients were quantified with this method. A comparison of the concentrations of DiAcSpm with those measured by a colloidal gold aggregation method demonstrated a correlation coefficient of 0.996, showing that the two methods were equally satisfactory. Analysis of the correlation between DiAcSpd or AcSpd and DiAcSpm, performed for the first time, indicated the usefulness of DiAcSpm as a urinary biomarker of cancer. During the course of this work, two simple methods for the preparation of α,ω-diacetylpolyamines were developed, and a possibility to separate and determine the concentrations of the two isomers, N¹-acetylspermidine and N⁸-acetylspermidine in AcSpd, was shown by tandem mass spectrometry (MS/MS).     

An immunocytochemical study on distinct intracellular localization of cathepsin E and cathepsin D in human gastric cells and various rat cells.

Immunocytochemical localization of two distinct intracellular aspartic proteinases, cathepsins E and D, in human gastric mucosal cells and various rat cells was investigated by immunogold technique using discriminative antibodies specific for each enzyme. Cathepsin D was exclusively confined to primary or secondary lysosomes in almost all the cell types tested, whereas cathepsin E was not detected in the lysosomal system. The localization of cathepsin E varied with different cell types. Microvillous localization of cathepsin E was found in the intracellular canaliculi of human and rat gastric parietal cells, rat renal proximal tubule cells, and the bile canaliculi of rat hepatic cells. The immunolocalization of each enzyme in gastric cells were essentially the same in humans and rats. In the gastric feveolar epithelial cells and parietal cells, definite immunolabeling for cathepsin E was observed in the cytoplasmic matrix, the cisternae of the rough endoplasmic reticulum, and the dilated perinuclear envelope. In rat kidney, cathepsin E was detected only in the proximal tubule cells, while cathepsin D was found mainly in the lysosomes of the distal tubule cells but not in those of the proximal tubule cells. These results clearly indicate the distinct intracytoplasmic localization of cathepsins E and D and suggest the possible involvement of cathepsin E in extralysosomal proteolysis that is related to specialized functions of each cell type.     

A missense mutation (Thr-6Pro) in the lysosomal acid lipase (LAL) gene is present with a high frequency in three different ethnic populations: Impact on serum lipoprotein concentrations

A frequent missense mutation (Thr-6Pro) found in the prepeptide of the lysosomal acid lipase (LAL) gene was analyzed in a cohort of 1003 randomly selected samples from Germany, Japan and Sardinia (Italy). Using the mutagenically separated polymerase chain reaction (MS-PCR), allele frequencies of 0.269, 0.238 and 0.245 were determined in the three populations, respectively. Statistical analysis showed a lack of association with a dyslipidemic phenotype in all three groups. Additionally, in a subgroup of 126 German individuals no association was observed between genotype and LAL activity. We conclude that this mutation appears to be a frequent LAL gene polymorphism causing no impaired function of the enzyme and no measurable dyslipidemia in the general population.This study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki