Purification of a unique glycoprotein that enhances phenol oxidase activity in scorpion (Heterometrus bengalensis) haemolymph.

A monomeric glycoprotein (SGP) of Mr 32,000 was isolated to purity from scorpion (Heterometrus bengalensis) haemolymph by (NH4)2SO4 fractionation, chromatofocusing and h.p.l.c. The homogeneity of SGP is confirmed by polyacrylamide-gel electrophoresis. SGP is soluble in 100%-satd. (NH4)2SO4 solution. Needle-shaped crystals of SGP were obtained in an aqueous environment. The glycan part of the molecule contains arabinose, which does not commonly occur in animal glycoproteins. Amino acid analysis demonstrated a preponderance of glycine, tyrosine and glutamic acid. SGP enhances phenol oxidase (EC 1.14.18.1) activity.     

Physiochemical studies on achatininH, a novel sialic acid-binding lectin.

The plasmid pEAP31 contains an alkaliphilic-Bacillus penicillinase gene and a colicin E1 kil gene. Escherichia coli HB101 carrying pEAP31 grown at high temperature released outer-membrane proteins, lipopolysaccharide and phosphatidylethanolamine into the culture medium. Concurrently, penicillinase that had accumulated in the periplasm of the organism was released from the cells. Phospholipase A1-A2 in the outer membrane was not activated in the organism. The results suggest that the release of accumulated periplasmic penicillinase from the producer cells was caused by partial disruption of the outer membrane mediated by the Kil peptide.     

Protection to testicular activity by a combination of 5-hydroxy L-tryptophan with a thiol compound in whole body gamma irradiated rats

Radiation induced changes in testicular activity were studied by estimating sialic acid content in plasma and testis and 17-ketosteroids in 24 hr urine samples of male Sprague Dawley rats following 8 Gy whole body gamma ray exposure with and without pretreatment with 2-aminoethylisothiuronium bromide hydrobromide (AET) or with a combination of 5-hydroxy L-tryptophan (5-HTP) and AET. Combination of 5-HTP with AET or AET alone in optimum radioprotecting dose has significantly modified the radiation damage to the testis.     

Abnormalities of the erythrocyte membrane in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a hematologic malignancy characterized by excessive growth of myeloid cells and their progenitors. The proportion of spectrin dimers compared to tetramers extracted from membranes at 4 degrees C, under low ionic strength conditions, increased in CML erythrocytes. These also displayed abnormal thermal sensitivity (between 45 and 46 instead of 49 degrees C). Crosslinking with the bifunctional reagent, dimethyl adipimidate (8.6 A) showed significant organizational modification of not only spectrin, but other cytoskeletal components such as ankyrin, bands 4.2 and 5. Enhanced concanavalin A (Con-A) agglutinability of CML erythrocytes also suggests altered topographic distribution of a functionally important membrane protein, band 3. The anion transport activities of erythrocytes from patients with CML and normal donors were comparable. In CML erythrocytes, significant reduction in the number of ankyrin-binding sites, present in the cytoplasmic domain of band 3, may lead to partial loss of cytoskeletal anchorage to the bilayer and account for their increased Con-A agglutinability and heat sensitivity and may lead to their premature removal from the circulation.     

Circular dichroism and fluorescence investigations on Vicia faba lectin-saccharide binding.

Vicia faba lectin contained 40-57% beta-conformation, 4-23% alpha-conformation along with random coil at pH 7.2 depending upon the analytical methods used. The percentage of beta-conformation increased with the addition of N-acetyl-D-glucosamine or methyl alpha-D-mannopyranoside. The structural transitions of V. faba lectin were affected by alkali at pH 9.6 and 10.6. Binding constants and free energy changes for the interaction between V. faba lectin and N-acetyl-D-glucosamine and methyl alpha-D-mannopyranoside were estimated at pH 7.2 using the c.d. and fluorescence methods.     

Vaginal hydrolysis of retinyl acetate: increase in plasma retinol and retinol binding protein in women with cervical dysplasias

We previously reported the clinical feasibility of a Phase I trial involving the topical administration of a RA gel applied cervicovaginally in women with mild or moderate cervical dysplasia. Now, we report hydrolysis and systemic absorption of the RA gel from the vagina. HPLC analysis of samples of residual gel obtained from the cervical canal after topical bolus application indicate that the RA undergoes prompt in vivo hydrolysis yielding retinol as a major metabolite. Venous blood samples of 41 subjects, who self-administered a RA gel, were analyzed for plasma retinol and RBP concentrations prior to and upon completion of a 7-day treatment course and upon return for follow-up examinations. An increase in both the concentrations of plasma retinol and RBP were detected after topical application of the RA gel. These elevated values receded after the gel administration was discontinued. No significant changes were observed in plasma retinol or RBP concentrations in placebo-treated subjects. The efficacy of RA as a chemopreventive agent in treating cervical dysplasias remains to be determined.     

Interactions of antisense DNA oligonucleotide analogs with phospholipid membranes (liposomes).

Antisense oligonucleotides have the ability to inhibit individual gene expression in the potential treatment of cancer and viral diseases. However, the mechanism by which many oligonucleotide analogs enter cells to exert the desired effects is unknown. In this study, we have used phospholipid model membranes (liposomes) to examine further the mechanisms by which oligonucleotide analogs cross biological membranes. Permeation characteristics of 32P or fluorescent labelled methylphosphonate (MP-oligo), phosphorothioate (S-oligo), alternating methylphosphonate-phosphodiester (Alt-MP) and unmodified phosphodiester (D-oligo) oligodeoxynucleotides were studied using liposomal membranes. Efflux rates (t1/2 values) at 37 degrees C for oligonucleotides entrapped within liposomes ranged from 7-10 days for D-, S- and Alt-MP-oligos to about 4 days for MP-oligos. This suggests that cellular uptake of oligonucleotides by passive diffusion may be an unlikely mechanism, even for the more hydrophobic MP-oligos, as biological effects are observed over much shorter time periods. We also present data that suggest oligonucleotides are unlikely to traverse phospholipid bilayers by membrane destabilization. We show further that MP-oligos exhibit saturable binding (adsorption) to liposomal membranes with a dissociation constant (Kd) of around 20nM. Binding appears to be a simple interaction in which one molecule of oligonucleotide attaches to a single lipid site. In addition, we present water-octanol partition coefficient data which shows that uncharged 12-15 mer MP-oligos are 20-40 times more soluble in water than octanol; the low organic solubility is consistent with the slow permeation of MP-oligos across liposome membranes. These results are thought to have important implications for both the cellular transport and liposomal delivery of modified oligonucleotides.     

Pyridoxal 5'-phosphate mediated inactivation of Escherichia coli DNA polymerase I: identification of lysine-635 as an essential residue for the processive mode of DNA synthesis

Inactivation of Escherichia coli DNA polymerase I by pyridoxal 5'-phosphate treatment results from its reactivity at multiple lysine residues. One of these residues, lysine-758, has been shown to be located at the substrate binding site in DNA polymerase I [Basu, A., & Modak, M. J. (1987) Biochemistry 26, 1704-1709]. We now demonstrate that lysine-635 is another important target of pyridoxylation; modification of this site results in decreased rates of DNA synthesis. Addition of template-primer with or without substrate deoxynucleoside triphosphate protects lysine-635 from pyridoxylation. Analysis of the initiation versus elongation phase of DNA synthesis by lysine-635-modified enzyme revealed that elongation of the DNA chain is severely affected by the lysine-635 modification. We therefore conclude that this lysine residue plays an important role in the processive mode of DNA synthesis by E. coli DNA polymerase I.     

Characterization of the activation of rat liver beta-glucosidase by sialosylgangliotetraosylceramide

We show that sialosylgangliotetraosylceramide (GM1) is a potent activator of delipidated (sodium cholate- and 1-butanol-extracted) lysosomal rat liver glucocerebroside:beta-glucosidase. Stimulation of 4-methylumbelliferyl-beta-D-glucopyranoside hydrolysis by the beta-glucosidase was markedly dependent upon the concentration of GM1 in the assay medium. Estimations of critical micellar concentration (CMC) performed fluorometrically using the dye N-phenylnaphthylamine revealed two CMC values of GM1 above 18 degrees C; the CMC of the primary micelles (3.32 microM) was temperature-independent whereas that of the secondary micelles decreased with decreasing temperature (17.2 and 10.8 microM at 37 and 20 degrees C, respectively). In the temperature range of 18-39 degrees C, beta-glucosidase activity increased sharply when the GM1 concentration was above the CMC of the secondary micelles. Although a heat-stable factor, purified from the spleen of a patient with Gaucher's disease, had a profound effect on the activation of beta-glucosidase by GM1, it decreased the CMC only slightly (14.8 versus 17.2 microM at 37 degrees C). The heat-stable factor (8 micrograms/ml) changed the shape of the activation curve from sigmoidal to hyperbolic, suggesting that the heat-stable factor permits beta-glucosidase to be activated by primary micelles or monomers. The results of gel filtration chromatography and sucrose gradient centrifugation in H2O and D2O revealed that the activation of beta-glucosidase by GM1 was associated with an increase in the size of the enzyme from 45,800 to 178,500 daltons and an increase in the partial specific volume from 0.697 to 0.740 ml/g. The active, reconstituted beta-glucosidase appears to consist of 50% protein and 50% ganglioside (56 molecules/178,500 g). Concentrations of GM1 below the CMC of secondary micelles increased the rate of inactivation of the enzyme by the irreversible inhibitor conduritol B epoxide at 37 degrees C, indicating that GM1 monomers or primary micelles do interact with the enzyme, even though they do not increase the rate of hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside by the enzyme.