Nematolysosomes (Elongate Lysosomes) in Rat Hepatocytes: Their Distribution, Microtubule Dependence, and Role in Endocytic Transport Pathway

The distribution of lysosomes in rat hepatocytes was examined by three-dimensional electron microscopy combined with acid phosphatase (ACPase) cytochemistry. In the 2-μm-thick sections observed under 200- or 1000-kV TEM, it was apparent that ACPase activity localized on elongate lysosomes (we refer to them as nematolysosomes) with a diameter of 70-100 nm and lengths of several micrometers, as well as spherical lysosomes and trans-Golgi cisternae. Though most spherical lysosomes were located within the pericanalicular region, nematolysosomes were widely distributed throughout the hepatocytes. Typically, it appeared that the nematolysosomes elongated from the subsinusoidal region to the pericanalicular-Golgi complex area and they frequently formed a network at the cell periphery along the sinusoidal front. Furthermore, the formation of nematolysosomes was independent of new protein synthesis, but highly dependent on the integrity of microtubules. After a 6-8 h colchicine treatment, nematolysosomes were shrunk and/or fragmented, becoming roughly spherical lysosomes scattered throughout the cells. Nematolysosomes recovered their normal profiles after 24 h due to the reversible effect of the drug on microtubules. When the hepatocytes were exposed to horseradish peroxidase (HRP) in vitro or in vivo, HRP was quickly sequestered in nematolysosome-like structures via pinocytosis from the sinusoidal surface and transported to an area near the Golgi complex. These findings raise the possibility that the nematolysosomes engage in microtubule-dependent transport of macromolecules from the sinusoidal circulation to the Golgi complex area.     

The GS-X Pump in Plant, Yeast, and Animal Cells: Structure, Function, and Gene Expression

This review addresses the recent molecular identification of several members of the glutathione S-conjugate (GS-X) pump family, a new class of ATP-binding cassette (ABC) transporters responsible for the elimination and/or sequestration of pharmacologically and agronomically important compounds in mammalian, yeast and plant cells. The molecular structure and function of GS-X pumps encoded by MRP, cMOAT, YCF1. and AtMRP genes, have been conserved throughout molecular evolution. The physiologic function of GS-X pumps is closely related with cellular detoxification, oxidative stress, inflammation, and cancer drug resistance. Coordinated expression of GS-X pump genes, e.g., MRP1 and YCF1, and -glutamylcystaine synthetase, a rate-limiting enzyme of cellular glutathione (GSH) biosynthesis, has been frequently observed.     

Influence of season on characteristics of epididymal and ejaculated semen in Meishan boars

Most of the epididymal spermatozoa collected in all the seasons examined maintained an ability to move progressively, had a cytoplasmic droplet in the distal site of the middle piece, and were morphologically normal. Reduced desire to mount a dummy was not observed during the experimental period. Characteristics of ejaculated semen were not significantly altered throughout the year. However, progressive motility and acrosomal integrity of spermatozoa ejaculated between July and September were more susceptible to storage at 4 degrees C than spermatozoa ejaculated during the other months and acrosomal integrity of spermatozoa ejaculated during the 3 months was to freezing-thawing. These results indicate that the reproductive activity of Meishan boars in Japan is only slightly influenced by season, but semen ejaculated during the summer is less suitable for storage than that ejaculated during the other seasons of the year.     

Adsorption mode of exo- and endo-cellulases from Irpex lacteus (Polyporus tulipiferae) on cellulose with different crystallinities.

The adsorption mode of two highly purified cellulases, exo- and endo-type cellulases, from Irpex lacteus (Polyporus tulipiferae) was investigated by using pure cellulosic materials with different crystallinity as substrates. Adsorption of the two enzymes on the substrates was found to fit the Langmuir-type adsorption isotherm. Maximum amount of adsorbed enzyme obtained from the Langmuir plots showed an inverse correlation to the crystallinity of the substrate with both enzymes, and this value of endo-type cellulase was less dependent on the degree of crystallinity of substrates than that of exo-type cellulase, whose isotherms reached saturation in the range of low enzyme concentrations. The two enzymes showed relatively high affinities for all the substrates and their affinities increased with increasing crystallinity, but this tendency was less marked with endo-type cellulase than with exo-type one. In addition, large negative values of free energy change were observed on the adsorption of both enzymes, and the values became more negative with increasing crystallinity. Consequently, both cellulases showed high adsorption on crystalline cellulose and the adsorption process became smoother with increasing crystallinity. The adsorption of the two types of cellulases was endothermic with an increase in entropy, especially for amorphous cellulose, suggesting the occurrence of water release from the substrates during enzyme adsorption. In addition, the changes in thermodynamic parameters (delta H, delta S, and delta G) in adsorption of exo-type cellulase were larger than in that of endo-type enzyme.     

Ovarian development in Meishan pigs

Ovaries collected from a total of 35 Meishan gilts of various ages were morphologically and histologically examined. Vesicular follicles were first observed in ovarian sections at 45 d of age. Simultaneously, protruding follicles appeared on the surface of the ovaries, and then ovarian weight increased rapidly with an increasing number of protruding follicles. About half of the gilts between 75 and 90 d of age had ovaries with corpora lutea, indicating that some of them had started estrous cycles before reaching 75 d of age. In Meishan gilts treated with exogenous gonadotropins, ovaries were stimulated as early as 45 d of age. These results suggest that the precocity of Meishan gilts may include the development of vesicular follicles in the ovaries at an early age.     

Development of a radioimmunoassay for human group-II phospholipase A2 and demonstration of postoperative elevation.

A radioimmunoassay (RIA) for the determination of human group-II phospholipase A2 (M-PLA2) has been developed. M-PLA2 was purified from human spleen. Monoclonal antibody (IgG) was prepared by fusion of splenic cells from immunized mice with M-PLA2 and the mouse myeloma cell line NS-1. The RIA was carried out by a single antibody method. The assay is sensitive (0.78 micrograms/l), reproducible and specific. In healthy individuals, the serum M-PLA2 concentration ranges from 1.4 to 4.2 micrograms/l, the average being 2.2 +/- 0.1 micrograms/l (mean +/- SE). Using the RIA, we found increased serum M-PLA2 in patients with various infections and malignant tumors. We also showed the postoperative transient elevation of serum M-PLA2 in cases without any infectious complications. The elevation was independent of the surgical procedure or site. The maximum serum M-PLA2 level was seen on the 2nd to 4th postoperative day. In these patients, the serum M-PLA2 and C-reactive protein levels were significantly correlated. The present study indicated that serum M-PLA2 is an acute phase reactant.     

35 kDa fragment of h-caldesmon conserves two consensus sequences of the tropomyosin-binding domain in troponin T

Using a tropomyosin-coupled affinity column, we have demonstrated a direct association between the chymotryptic 35 kDa fragment of h-caldesmon, which is located at the C-terminal of the parent molecule, and gizzard tropomyosin. We have subsequently determined the nucleotide sequence of cDNA clones encoding the 35 kDa fragment from the cDNA library prepared from chick embryo gizzards, and have deduced the amino acid sequence. Calculating from the predicted sequence, the 35 kDa fragment is composed of 306 amino acid residues. In agreement with the tropomyosin-binding ability, the 35 kDa fragment conserves two consensus sequences of the tropomyosin-binding domain in troponin T. These results suggest that the 35 kDa fragment of h-caldesmon, at least in part, has a common property to the striated muscle troponin T.     

The primary structure of a membrane-associated phospholipase A2 from human spleen

The complete primary structure of membrane-associated phospholipase A2 purified from a human splenic membrane fraction was determined by sequence analysis of the peptides generated by lysyl endopeptidase and Staphylococcus aureus V8 protease cleavage. The enzyme consists of 124 amino acid residues corresponding to a molecular weight of 13,904. The primary structure reveals the characteristics of Group II phospholipases A2 and a large ratio of basic amino acid residues to acidic ones, that ratio being 3.4 : 1.     

Substrate specificities of exo- and endo-type cellulases in the hydrolysis of beta-(1----3)- and beta-(1----4)-mixed D-glucans

An exo-type cellulase (Ex-1) was extracted from Irpex lacteus (Polyporus tulipiferae) and purified essentially to homogeneity. This cellulase attacked cellulosic substrates in an exo-wise fashion to produce almost exclusively cellobiose. In contrast, Ex-1 was found to attack beta-glucans having beta-(1----3)- and beta-(1----4)-mixed linkages in a way similar to an endo-type cellulase. The products formed from barley glucan by Ex-1 were 3(2)-O-beta-D-cellobiosyl-cellobiose much greater than 3(2)-O-beta-D-glucosyl-cellobiose greater than cellobiose much greater than or equal to cellotriose much greater than glucose in the early stage, but no laminaribiose was produced. An endo-type cellulase (En-1) obtained from the same fungus also hydrolyzed beta-glucans but in a typical endo-wise fashion and the products from barley glucan were 3(2)-O-beta-D-glucosyl-cellobiose much greater than 3(2)-O-beta-D-cellobiosyl-cellobiose greater than cellobiose much greater than laminaribiose; no glucose or cellotriose was produced. Thus, it seems likely that En-1 can attack any intramolecular linkage of beta-glucan, while Ex-1 requires the presence of at least cellobiosyl residues adjacent to a beta-(1----3)-D-linked glucosyl residue. This finding, together with the mode of hydrolysis of cellulosic substrates by Ex-1, suggests that the stereochemical structure of successive beta-(1----4)-cellobiosyl residues inserted by beta-(1----3)-D-glucosidic linkage is permissible in the action of Ex-1, although this enzyme prefers the beta-(1----4)-linked cellobiosyl sequence.     

A novel phage genome integrated into a plasmid in Bacillus thuringiensis strain AF101

Bacillus thuringiensis strain AF101 possesses a single plasmid (pAF101) with a molecular size of 42 MDa (69 kb). During plasmid curing experiments in strain AF101, we found that a phage (J7W-1) was induced by ethidium bromide treatment. Moreover, the phage genome (48 kb) hybridized only with pAF101 on a Southern blot of the DNA of a cleared lysate prepared from strain AF101. Comparison of the restriction patterns of pAF101 and J7W-1 phage DNA revealed that pAF101 contains not only the entire phage DNA but also a plasmid-specific DNA region. These results indicate that the J7W-1 genome has been stably integrated into pAF101 in strain AF101. Integration of the J7W-1 genome into a plasmid was also observed after phage infection of the type strain of B. thuringiensis subsp. israelensis.