Sse8387I, a useful eight base cutter for mammalian genome analysis (influence of methylation on the activity of Sse8387I).

To develop restriction enzymes that are useful for genome analysis, we previously performed screening and isolated Sse8387I from Streptomyces sp. strain 8387. Sse8387I is a restriction enzyme that recognizes 5'-CCTGCA/GG-3' and cleaves DNA at the site shown by the diagonal (Nucleic Acid Res., 18, 5637-5640). The present study evaluated the effects of methylation that is important when Sse8387I is used for genome analysis. Sse8387I lost cleavage activity after methylation of adenine or methylation of cytosine at any site in the recognition sequence. However, the recognition sequence of Sse8387I contains no CG sequence, which is the mammalian methylation sequence. In addition, we evaluated the effects of methylation of CG at sites other than the recognition sequence. The cleavage activity of Sse8387I was maintained even when CG sequences were present immediately before or after, or near the recognition sequence, and cytosine was methylated. These results suggest that CG methylation does not affect the cleavage activity of Sse8387I. Therefore, Sse8387I seems to be very useful for mammalian genome analysis.     

Establishment and characterization of a clonal human T-cell line, MKB-1 derived from a patient with acute myeloblastic leukemia

A human T-cell line, designated as MKB-1, was established by cloning procedures in a suspension culture from a peripheral blood of a 17-year-old female patient with acute myeloblastic leukemia. The immunological marker profile of MKB-1 indicated that unlike a myeloid phenotype of the original leukemic cells, the cells were positive for CD3 (both cell surface and cytoplasm), T cell receptor (TcR) alpha/beta heterodimer, CD4, CD5, CD7, CD10, CD57 (Leu7), SN-1 and the cytoplasmic TcR beta chain. These findings indicate the T cell nature of the established cells. Terminal deoxynucleotidyl transferase (TdT) was also detected in 60%. We did not detect markers of human myeloid and B cell associated antigens, HLA-class II or immunoglobulin chains. Cytogenetic study revealed that the MKB-1 cells had a female hypo-tetraploid karyotype with chromosomal abnormalities including a translocation between chromosomes 10 and 14. The breakpoint of chromosome 14 of this translocation, 14q11.2, is known to be the location of TcR alpha and delta genes; t(10; 14) (q26; q11.2) is a variant type of a T cell neoplasm-associated translocation, t (10; 14) (q24; q11.2). The MKB-1 cell line is unusual in that its T cell characteristics are phenotypically and cytogenetically distinct from the original myeloid leukemia cells.     

Sse8387I, a new type-II restriction endonuclease that recognizes the octanucleotide sequence 5''-CCTGCAGG-3''.

A type II restriction endonuclease designated Sse8387I was partially purified from Streptomyces sp. 8387. This enzyme cleaved adenovirus 2 DNA at three sites, lambda phage DNA at five sites, and pUC18 and M13mp18 RF DNA at one site each, but did not cleave the DNAs from pBR322, SV40, or phi X174. Sse8387I recognized the octanucleotide sequence 5'-CCTGCA decreases GG-3', cleaving where shown by the arrow. Sse8387I is the first restriction endonuclease to be reported that recognizes an octanucleotide sequence consisting of all four nucleotides, G, A, T, and C. The frequency of occurrence of Sse8387I sites within sequenced regions of primate genomes was 2.4 times that of NotI sites.     

Phenotyping of malignant hematopoietic cells. Analysis of 1200 cases of leukemia-lymphoma

1255 cases of leukemia-lymphoma were tested between 1972 and 1984 by multiple marker analysis. Routine leukemia phenotyping was performed using standard morphological and cytochemical techniques in combination with clinical and histo-pathological information; the main emphasis was put on immunological surface marker analysis using erythrocyte rosette assays, TdT and a large panel of poly- and monoclonal antibody tests. The 1255 cases were divided into these major types and subtypes: 349 cases of ALL and related immature T- and Burkitt-lymphomas (cALL, pre B-ALL, B-ALL and Burkitt-lymphomas, T-ALL and immature, mostly leukemic T-lymphomas, Null-ALL), 454 cases of mature T- and B-cell malignancies (T-CLL, mycosis fungoides, Sezary-syndrome, T-lymphomas, B-CLL, hairy cell leukemia, multiple myeloma, B-lymphomas), 263 cases of acute myeloid leukemias (AML, AMMoL/AMoL), 182 cases of chronic myeloid leukemias (CML in chronic phase, CMoL, CML in blast crisis), 6 cases of erythroleukemia and 1 case of megakaryoblastic leukemia. A simplified classification scheme which has been used in our laboratories is presented. Phenotyping is of diagnostic, prognostic and therapeutic relevance, most evidently for patients with ALL. Routine leukemia phenotyping should be performed with highly standardized techniques and reagents and by combining information from several fields in the multiple marker analysis. New areas of leukemia research might become very useful for the routine procedure of phenotyping.     

Simultaneous occurrence of myelomonocytic leukemia and multiple myeloma: involvement of common leukemic progenitors and their developmental abnormality of "lineage infidelity".

We investigated the origin of leukemic progenitors in a case of the simultaneous occurrence of myelomonocytic leukemia and multiple myeloma (IgG-kappa). At presentation, myeloperoxidase and nonspecific esterase-positive myelomonocytic cells had proliferated up to 12.2 x 10(9)/liter in the peripheral blood. Bone marrow cell differentials revealed the coexistence of myelomonocytic cells (30%) and atypical plasmacytoid cells (26%). Myelomonocytic cells in peripheral blood expressed both myeloid antigens (CD11b, CD13, CD14, CD15, CD33) and T/B-lymphoid antigens (CD2, CD4, CD5, CD7, CD10, PCA-1). Bone marrow mononuclear cells (BMMC) could be divided into PCA-1 strongly positive and PCA-1 weakly positive populations, which were considered to represent myeloma cells and myelomonocytic cells, respectively; the former were CD2-positive (CD2+), CD14-, and CD15-, whereas the latter were CD2+, CD14+, and CD15+. Immunohistochemical analysis revealed that, in addition to plasmacytoid cells, a minority of myelomonocytic cells showed a positive reaction for IgG staining, and production of IgG was observed in the culture supernatant of CD14+ myelomonocytic cells in peripheral blood. Southern blot analysis revealed the presence of two identical rearrangement bands of immunoglobulin heavy chain gene in both BMMC containing myeloma cells and myelomonocytic cells and CD14+ myelomonocytic cells in peripheral blood. In a long-term methylcellulose assay, peripheral blood mononuclear cells produced large compact colonies consisting of macrophages and IgG+ plasmacytoid cells (M phi/P colonies), while BMMC produced a different type of colonies consisting of CD14+ myelomonoblasts, macrophages, and IgG+ plasma cells (Mb/M phi/P colonies) in addition to M phi/P colonies. Recloning experiments showed that primary Mb/M phi/P colonies gave rise to both secondary M phi/P and Mb/M phi/P colonies. These observations strongly suggest that common leukemic progenitors provide both myeloma and myelomonocytic leukemia cells, and the mechanism of "lineage infidelity" is probably involved in the development of their "bilineal" differentiation.     

Effects of low dose L-triiodothyronine administration on mental, behavioural and thyroid states in elderly subjects

Decreased serum T3 concentrations in elderly subjects and their possible relationship with the development of dementia have been indicated. To see the effects of a passive increase in the serum T3 concentration, low dose T3 administration was undertaken. Forty-four subjects from 65 to 93 years of age (average 81.0 +/- 7.8) were divided into 2 groups. The grade of dementia was determined by Hasegawa's dementia rating scale (DR score). In 22 subjects, 25 micrograms per day of T3 was administered for 4 W, while the control group was given a placebo. The DR score was measured before and immediately after the study. Changes in behaviour were monitored in a double-blind fashion. The administration of T3 induced a 0.65 nmol/l increase in serum T3 in 2 W and 0.36 nmol/l in 4 W. These T3 increases were not associated with significant changes in the DR score but 7 of 22 subjects showed apparent improvement in behaviour. TSH was suppressed to less than 1 mU/l in 2 W and then slightly increased by the 4th week, but T4, rT3 and fT4 all showed significant and progressive decreases. The DR score after T3 correlated significantly with the rT3/T4 ratio (before T3: -0.55, changes: +0.50) and also with changes in rT3 (r = 0.49). In conclusion, T3 administration to the elderly subjects was associated with behavioural improvement in some individuals, but the intellectual ability as assessed by the DR score in those with low T3 or elevated rT3 were hardly improved by passive T3 elevation.     

Changes in fish assemblage structure with variability of flow in two different channel types

Despite the fact that flow and channel morphology are two critical factors that need to be taken into account when considering the conservation or restoration of river environments, there are no reports on the response of fish assemblages to changes involving both variables in combination. This study examined the response of fish assemblages to artificially induced changes in flow discharge in an experimental stream that had simple (a glide reach) and complex (pool–riffle reach) morphologies. When the flow of the glide reach was increased, the fish assemblage structure changed from one dominated by the demersal species Misgurnus anguillicaudatus and Cobitis sp. to one dominated by the water-column species Zacco platypus and Gnathopogon elongatus. In the pool–riffle reach, the fish assemblage structure changed from one dominated by Cobitis sp. and Z. platypus to one dominated by Plecoglossus altivelis altivelis. When the flow discharge was low, the fish assemblages of both reach types resembled each other in being dominated largely by demersal species, but when flow was increased these similarities faded and distinct assemblages emerged. In the glide reach, increasing the flow volume caused a linear increase in both water depth and velocity and a gradual increase in the diversity of water-column species, their relative abundance, and size. The number of Z. platypus increased fivefold whereas the number of M. anguillicaudatus decreased to less than a quarter of their original number. In the pool–riffle reach, the number of P. altivelis altivelis grew conspicuously although increased flow produced no clear increase in depth or velocity. Calculation of the availability of physical habitat environments suitable for Z. platypus and P. altivelis altivelis in each reach type indicated that preferable habitat for both species was more available in the glide reach. The lower abundance of P. altivelis altivelis in the glide reach was attributed to the relatively low availability of algal food resource due to the sand-predominated substrate whereas pebbles predominated in the pool–riffle reach providing good conditions for algal growth. Our findings suggest the need for a framework for considering environmental flow that takes into account variables such as channel morphology and food resource conditions.     

Cation-dependent nutrient transport in shrimp digestive tract

Purified epithelial brush border membrane vesicles (BBMV) were produced from the hepatopancreas of the Atlantic White shrimp, Litopeneaus setiferus, using standard methods originally developed for mammalian tissues and previously applied to other crustacean and echinoderm epithelia. These vesicles were used to study the cation dependency of sugar and amino acid transport across luminal membranes of hepatopancreatic epithelial cells. ³H-d-glucose uptake by BBMV against transient sugar concentration gradients occurred when either transmembrane sodium or potassium gradients were the only driving forces for sugar accumulation, suggesting the presence of a possible coupled transport system capable of using either cation. ³H-l-histidine transport was only stimulated by a transmembrane potassium gradient, while ³H-l-leucine uptake was enhanced by either a sodium or potassium gradient. These responses suggest the possible presence of a potassium-dependent transporter that accommodates either amino acid and a sodium-dependent system restricted only to l-leucine. Uptake of ³H-l-leucine was significantly stimulated (P < 0.05) by several metallic cations (e.g., Zn²⁺, Cu²⁺, Mn²⁺, Cd²⁺, or Co²⁺) at external pH values of 7.0 or 5.0 (internal pH 7.0), suggesting a potential synergistic role of the cations in the transmembrane transfer of amino acids. ³H-l-histidine influxes (15 suptakes) were hyperbolic functions of external [zinc] or [manganese], following Michaelis–Menten kinetics. The apparent affinity constant (e.g., K _m) for manganese was an order of magnitude smaller (K _m = 0.22 μM Mn) than that for zinc (K _m = 1.80 μM Zn), while no significant difference (P > 0.05) occurred between their maximal transport velocities (e.g., J _max). These results suggest that a number of cation-dependent nutrient transport systems occur on the shrimp brush border membrane and aid in the absorption of these important dietary elements.     

Assessment of a high-throughput screening methodology for the measurement of purified UCP1 uncoupling activity

Three mitochondrial uncoupling proteins (UCP1, 2, 3) have been described. The proton transport activity of UCP1 triggers mitochondrial uncoupling and thermogenesis but the roles of UCP2 and UCP3 remain debated. Accordingly, compounds able to finely control the proton permeability of the mitochondrial inner membrane where and when needed may have enormous practical consequences. Using purified hamster brown adipose tissue UCP1 reconstituted in liposomes, we describe herein a robust assay allowing the measurement of this artificial membrane conductance to protons in a format compatible with high-throughput screening. The assay was initially developed with a known chemical protonophore in an aproteic system. Then, using the proteolipid reconstituted UCP1 preparation, we assessed the assay with known modulators of UCP1, particularly retinoic acid and guanosine 5'-triphosphate. The system was developed for a 96-well plate format. We then exemplified its use by generating primary data on a set of compounds screened in this system. These primary data will open new routes for the search of candidate compounds that will help biochemical studies on UCPs.