A novel galactosyl-binding lectin from the plasma of the blood clam,Anadara granosa (L) and a study of its combining site

Control Analysis has been carried out in the first steps of a rat liver glycolytic system. Attention has been focused on the effect of several glucose concentrations on the control, particularly regarding the role of glucokinase. From kinetic studies of the whole metabolic system we have obtained information on the flux variation under different glucose concentrations. This information together with the kinetics of glucokinase has allowed us to calculate Flux Control and Elasticity Coefficients for glucokinase and the Response Coefficient of the system with respect to glucose. The changes in of the value of Flux Control Coefficients demonstrates that in conditions of low glucose concentration, glucokinase is the main enzyme in controlling the flux through the pathway, but at high glucose concentration the control moves to phosphofructokinase. Next, we have compared our results with those obtained with the shortening and titration method, previously described (Torres, N.V., Mateo, F., Mélendez-Hevia, E. and Kacser, H., (1986) Biochem. J. 234, 169–174; Torres, N.V. and Meléndez-Hevia, E. 1991. Molec. Cell. Biochem. 101, 1–10). Furthermore, from knowledge of the enzyme kinetics of the system we have been able to build a model of the pathway that allows us computer similation of its behavior and calculation of the Flux Control Coefficient profile at different glucose concentrations. By the three methods the results correlate, supporting the use of the pathway substrate as external modulator of the metabolic system as a tool for practical application of Control Analysis.     

Selection of Lactobacillus acidophilus strains for use in “acidophilus products”

Recent studies by DNA-DNA hybridization revealed that strains now designated as L. acidophilus, can be divided into several groups and only one group should be classified as L. acidophilus. We studied several phenotypic characteristics in representative strains from the six DNA-homology groups of L. acidophilus. No group specific pattern was observed among the strains for fermentation of eight carbohydrates, growth at 15 and 45°C, resistance to 0.2% oxgall, lysis by lysozyme or sensitivity to 17 antibiotics. However, some differences among groups were observed in -galactosidase (-gal) activity and surface layer (s-layer) protein. Strains in B1 do not have a s-layer or -gal while B2 strains also lack a s-layer but do possess -gal. All strains in groups A1, A2, A3 and A4, capable of growing in lactose, have -gal activity and also have a s-layer composed of protein subunits of different molecular weights (MW). Strains in A1 homology group have a s-layer with 46 Kd protein subunits while strains in other A groups have s-layer protein subunits that varied in MW within each group. On the basis of these two traits several isolates of unknown homology groups have been tentatively placed in A1, B1 or B2 groups. L. acidophilus from A1 group showed strain variation in -gal specific activity and rate of acid production and growth. For use in dietary adjuncts, L. acidophilus strains should be selected for these three and other desirable traits. They should be maintained and grown in media containing lactose.     

Antibody directed targeting of methotrexate-containing small unilamellar vesicles

Summary The potential of antibody-linked SUVs containing MTX in anticancer therapy was investigated. The SUVs, mean diameter 50±20 nm, were prepared by probe sonication of MTX-containing MLVs and were covalently linked either to a RAMG or NRG. After incubation with M21 melanoma cells for 2 h, RAMG-linked SUVs showed 2 and 4 times more binding than NRG-linked MTX-containing SUVs or MTX-containing SUVs unlinked to any Ig. Furthermore, on incubating M21 melanoma cells with RAMG-linked ³H MTX-containing SUVs for 2, 4, and 8 h at 4° C or 37° C, a higher radioactivity was associated with cells at 37° C than at 4° C. Membrane immunofluorescence revealed aggregation of and cap formation by RAMG-linked SUVs after 2 h (37° C) and endocytosis at 4 and 8 h at 37° C. Electron microscopic and autoradiographic studies confirmed aggregation of ³H MTX-containing SUVs around and on the surface of M21 cells. Electron microscopy also revealed these SUVs inside invaginations of and under the plasma membrane of melanoma cells. A colony inhibition assay showed that RAMG-linked, MTX-containing SUVs were 60 times, 8 times, and 4.5 times more growth inhibitory than free MTX, NRG-linked MTX-containing SUV, and MTX-containing SUVs unlinked to any Ig, but not toxic to a human kidney cancer line (that did not react with RAMG). Abbreviations used: DPPC, DL- -dipalmitoyl phosphatidylcholine; DTT, dithiothreitol; MTX, methotrexate; (MTX)SUV or MLV, MTX-containing SUV or MLV; MLV, multilamellar vesicle; NRG, normal rabbit immunoglobulin G; RAMG, rabbit antimelanoma IgG; SA, stearylamine; SPDP, N-succinimidy1-3-(2-pyridyldithio)propionate; SUV, small unilamellar vesicle; CHOL, cholesterol; LUV, large unilamellar vesicle; Ig, immunoglobulin; PDP-SA, N-[3-(2-pyridyldithio)-propinyl]stearylamine     

Regression of human melanoma xenografts in nude mice injected with methotrexate linked to monoclonal antibody 225.28 to human high molecular weight-melanoma associated antigen

Summary Intravenous injections into nude mice of 5 mg/kg methotrexate (MTX) linked to the antibody to human high molecular weight-melanoma associated antigen (HMW-MAA), monoclonal antibody (mAb) 225.28, an IgG_2a, on days 1, 4, 7, 10 and 14, starting 24 h after subcutaneous inoculation of 2 × 10⁶ cultured human M21 melanoma cells inhibited mean tumor volume by 90% on day 14 and by 65% on day 50 after the beginning of the treatment. Injections of equimolar amounts of free MTX and MTX linked to normal mouse IgG or to an isotypematched myeloma protein did not inhibit tumor growth significantly. MTX linked to mAb 225.28 did not inhibit the xenograft of a subline of human melanoma cell line M21 without detectable expression of HMW-MAA. In a clonogenic assay, the MTX-225.28 conjugate was three times more potent in inhibiting the growth of M21 melanoma cells than free MTX, but did not inhibit the growth of kidney carcinoma cells Caki-1, which do not express high-M _r MAA. In contrast, MTX linked to the mAb DAL K29, reacting with kidney carcinoma cells Caki-1, inhibited their growth but did not affect that of melanoma cells. M21 melanoma cells isolated from the residual tumor of a mouse treated with the MTX-225.28 conjugate did not differ in their reactivity with mAb 225.28 and in their sensitivity to MTX when compared with M21 cells from an untreated mouse.     

Electrical conductivity of bilayer lipid membranes in electrolytic solution

The electrical conductivity of bilayer lipid membranes (BLM) of oxidized cholesterol has been measured separately in bathing solutions of sodium sulphate, sodium chloride, sodium bromide, sodium iodide and also in bathing solutions of iodine and iodine containing these salts. An attempt has been made to explain the conduction of electric current across the membranes.     

Characterization of the Lactobacillus helveticus CNRZ32 pepC gene.

Sequence analysis of the aminopeptidase C gene (pepC) from Lactobacillus helveticus CNRZ32 identified a 1,332-nucleotide open reading frame coding for a polypeptide with motifs characteristic of cysteine proteinases. Homology to the pepC gene appears to be widely distributed among lactic acid bacteria.     

Rat Brain 3-Hydroxy-3-Methylglutaryl-CoA Reductase: Subcellular Distribution and Cold Lability

Abstract: Data are provided indicating that the rat brain 3-hydroxy-3-methyl-glutaryl-CoA reductase is similar to the enzyme from other tissues as far as diurnal rythmicity, cold lability and half-life measurements at 0°C are concerned. The enzyme activity in the brain decreased with age of the animals. Subcellular fractionation studies demonstrate that while 77% of the activity was associated with the microsomal fraction, 19% of the enzyme activity was recovered in the mitochondrial fraction. The possible function of such a mitochondrially located 3-hydroxy-3-methylglutaryl-CoA reductase in rat brain is discussed.     

Some structural features of the d-glucan from the seed of Mirabilis jalapa

The cotyledon of the seed of Mirabilis jalapa was found to contain a d-glucan. Methylation, periodate oxidation, and graded and enzymic hydrolysis studies were conducted to elucidate its structure. For every 38 d-glucosyl residues therein, 34 are (1→4)- and 3 are (1→3)-linked; the d-glucosyl unit at the branch point is linked through O-1, O-2, and O-4. In some places in the chain, there are at least three (1→3)-linked d-glucosyl residues in a sequence. Both α- and β-d-glucosidic linkages are present in the polysaccharide, the former preponderating. The d-glucan gave with iodine a faint blue color that had λ_max 420 nm.     

Liposome formation of egg lecithin and its interaction with iodine

The sonicated dispersion of egg lecithin (phosphatidylcholine) in water forms 1:1 molecular complex with iodine, when its concentration is above 1.6 X 10(-5) M. The thermodynamic and spectrophotometric properties of this complex have been determined. The thermodynamic values are: K (25 degrees C) = 1.6 X 10(3) 1 X mol-1, delta G degrees = -18.4 KJ X mol-1, delta H degrees = -27.4 KJ X mol-1 and delta S degrees = -30.0 J X mol-1 X deg-1. The complex shows two absorption bands: one at 293 nm, which is the charge transfer band and the other at 370 nm, which is the blue shifted visible iodine band at 460 nm in water.