The mapping and reconstitution of a conformational discontinuous B-cell epitope of HIV-1

A method for the discovery of the structure of conformational discontinuous epitopes of monoclonal antibodies (mAbs) is described. The mAb is used to select specific phages from combinatorial phage-display peptide libraries that in turn are used as an epitope-defining database that is applied via a novel computer algorithm to analyze the crystalline structure of the original antigen. The algorithm is based on the following: (1) Most contacts between a mAb and an antigen are through side-chain atoms of the residues. (2) In the three-dimensional structure of a protein, amino acid residues remote in linear sequence can juxtapose to one another through folding. (3) Tandem amino acid residues of the selected phage-displayed peptides can represent pairs of juxtaposed amino acid residues of the antigen. (4) Contact residues of the epitope are accessible to the antigen surface. (5) The most frequent tandem pairs of amino acid residues in the selected phage-displayed peptides can reflect pairs of juxtaposed amino acid residues of the epitope. Application of the algorithm enabled prediction of epitopes. On the basis of these predictions, segments of an antigen were used to reconstitute an antigenic epitope mimetic that was recognized by its original mAb.     

Adipose tissue sensitization to insulin induced by troglitazone and MEDICA 16 in obese Zucker rats in vivo

The putative role played by insulin sensitizers in modulating adipose tissue lipolysis in the fasting state was evaluated in obese conscious Zucker rats treated with troglitazone or beta,beta'-tetramethylhexadecanedioic acid (MEDICA 16) and compared with nontreated lean and obese animals. The rates of appearance (R(a)) of glycerol and free fatty acid (FFA), primary intra-adipose reesterification, and secondary reuptake of plasma FFA in adipose fat were measured using constant infusion of stable isotope-labeled [(2)H(5)]glycerol, [2,2-(2)H(2)]palmitate, and radioactive [(3)H]palmitate. The overall lipolytic flux (R(a) glycerol) was increased 1.7- and 1.4-fold in obese animals treated with troglitazone or MEDICA 16, respectively, resulting in increased FFA export (R(a) FFA) in the troglitazone-treated rats. Primary intra-adipose reesterification of lipolysis-derived fatty acids was enhanced twofold by insulin sensitizers, whereas reesterification of plasma fatty acids was unaffected by either treatment. Despite the unchanged R(a) FFA in MEDICA 16 or the increased R(a) FFA induced by troglitazone, very low density lipoprotein production rates were robustly curtailed. Total adipose tissue reesterification, used as an estimate of glucose conversion to glyceride-glycerol, was increased 1.9-fold by treatment with the insulin sensitizers. Our results indicate that, in the fasting state, insulin sensitizers induce, in vivo, a significant activation rather than suppression of adipose tissue lipolysis together with stimulation of glucose conversion to glyceride-glycerol.     

Estrogen regulates cytokine production and apoptosis in PMA-differentiated,macrophage-like U937 cells

We have investigated the effects of sex steroids, estradiol (E2), and testosterone (T) on the synthesis of tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) in phorbol-myristate-acetate (PMA)-differentiated human monoblastic U937 cells. The ability of both hormones to modulate the viability and programmed cell death of macrophage-like PMA-differentiated U937 cells was also inspected. E2 increased TNF-alpha synthesis, whereas T had no effect on the production of this cytokine. The combination of E2 and its antagonist tamoxifen or ICI-182,789 completely abolished the induction of TNF-alpha, while combination of T and its antagonist Casodex (CSDX) did not significantly affect TNF-alpha production by U937 cells. Exposure of cells to E2 resulted in a dose-dependent decrease of IL-10 synthesis, while again T did not show any detectable effect. In addition, E2 induced a significant increase of apoptosis in macrophage-like U937 cells and this increase was inhibited by the simultaneous addition of either tamoxifen or ICI-182. In contrast, T alone or in combination with CSDX did not modify apoptotic rates of U937 cells. This evidence, taken together, suggests that estrogens, but not androgens, exert a pro-inflammatory action through the modulation of TNF-alpha and IL-10, and regulate the immune effector cells by the induction of programmed cell death.     

Human plasma uridine diphosphate galactose-glycoprotein galactosyltransfertase. Purification, properties and kinetics of the enzyme-catalysed reaction.

The soluble galactosyltransferase of human plasma catalysed the transfer of galactose from UDP-galactose to high- and low-molecular-weight derivatives of N-acetylglucosamine, forming a beta-1-4 linkage. The enzyme was purified by using (NH4)2SO4 precipitation and affinity chromatography on an alpha-lactalbumin-Sepharose column. The galactosyltransferase was maximally bound to this column in the presence of N-acetylglucosamine, and the enzyme was eluted by omitting the amino sugar from the developing buffer. The molecular weight of the enzyme was estimated to be 85000 by gel filtration. The assay conditions for optimum enzymic activity was 30 degrees C and pH7.5. Mn2+ ion was found to be an absolute requirement for transferase activity. The Km for Mn2+ was 0.4 mM and that for the substrate, UDP-galactose, was 0.024 mM. The Km for the acceptors was 0.21 mM for alpha1-acid glycoprotein and 3.9 mM for N-acetylglucosamine. In the presence of alpha-lactalbumin, glucose became a good acceptor for the enzyme and had a Km value of 2.9 mM. Results of the kinetic study indicated that the free enzyme reacts with Mn2+ under conditions of thermodynamic equilibrium, and the other substrates are added sequentially.     

Determination of taste threshold

A linear inverse relation links the HCl solution molarity to both the absolute HCl solution intake, and the ratio of the HCl solution to the total fluid intake. The last algebraic relation allows the taste threshold to be estimated.     

Rat intestinal brush border membrane dipeptidyl-aminopeptidase IV: kinetic properties and substrate specificities of the purified enzyme

The kinetic properties and substrate specificities of dipeptidyl-aminopeptidase IV (EC 3.4.14.—) detergent-solubilized and purified from the brush border membrane of rat small intestinal mucosal cells were investigated. Kinetic analysis of purified dipeptidyl-aminopeptidase IV was carried out with a variety of oligopeptides and β-napthylamide derivatives as substrates. In general, peptides with proline penultimate to the amino terminus (XPro, X= amino acid) are more favored substrates while those with alanine (XAla) are hydrolyzed at a slower rate. There is some activity toward substrates having leucine at both the penultimate position and amino terminus (LeuLeu). The activity of the purified enzyme toward GlylProβ-napthylamide derivative is maximal at pH 8.4 in Tris-HCl buffer, with an activation energy of 7.98 kcal/mol. There is no requirement for metal ion. The ability of various dipeptides to inhibit Gly-l-Pro-β-napthylamide derivative hydrolysis was used to determine the binding specificity of the enzyme for the amino-terminal amino acid. These data show that a free amino acid group is necessary for enzymatic activity and increased hydrophobicity of the amino acid at the amino terminus enhances binding.     

Physiological and morphological effects of genetic alterations leading to a reduced synthesis of UDP-glucose in Saccharomyces cerevisiae

Yeast cells lacking UDP-Glc pyrophosphorylase (UGPase) encoded by UGP1 are not viable. Two strategies were developed to drastically reduce the intracellular concentration of UDP-Glc in order to study the consequences of this metabolic engineering on physiology and morphology. Firstly, UGP1 was placed under the strongly regulatable THI4 promoter. This resulted in a 95% reduction of UGPase activity in the presence of thiamine. The phenotypic effects of this reduction were slightly stronger than those of glucose on the GAL10/CYC1-UGP1 gene fusion [Daran et al. (1995) Eur. J. Biochem. 230, 520–530]. A further reduction of flux towards UDP-Glc was achieved by deletion of the two phosphoglucomutase genes in the ugp1 conditional strain. The growth of this new mutant strain was hardly affected, while it was extremely sensitive to cell wall interfering drugs. Surprisingly, UDP-Glc levels were reduced only by 5-fold, causing a proportional decrease in both glycogen and β-glucans. Taken altogether, these results indicate that a few percent of enzymatic activities leading to the formation of UDP-Glc appears sufficient to provide the UDP-Glc demands required for cell viability, and that the loss of function of UGP1 is lethal mainly because of the inability of yeast cells to properly form the cell wall.     

The planar cell polarity protein Strabismus promotes Pins anterior localization during asymmetric division of sensory organ precursor cells in Drosophila

Cell fate diversity is generated in part by the unequal segregation of cell-fate determinants during asymmetric cell division. In the Drosophila bristle lineage, the sensory organ precursor (pI) cell is polarized along the anteroposterior (AP) axis by Frizzled (Fz) receptor signaling. We show here that Fz localizes at the posterior apical cortex of the pI cell prior to mitosis, whereas Strabismus (Stbm) and Prickle (Pk), which are also required for AP polarization of the pI cell, co-localize at the anterior apical cortex. Thus, asymmetric localization of Fz, Stbm and Pk define two opposite cortical domains prior to mitosis of the pI cell. At mitosis, Stbm forms an anterior crescent that overlaps with the distribution of Partner of Inscuteable (Pins) and Discs-large (Dlg), two components of the anterior Dlg-Pins-Galphai complex that regulates the localization of cell-fate determinants. At prophase, Stbm promotes the anterior localization of Pins. By contrast, Dishevelled (Dsh) acts antagonistically to Stbm by excluding Pins from the posterior cortex. We propose that the Stbm-dependent recruitment of Pins at the anterior cortex of the pI cell is a novel read-out of planar cell polarity.     

Postpartum ovarian follicular dynamics in primiparous and pluriparous Mediterranean Italian buffaloes (Bubalus bubalis)

The objective of this study was to monitor ovarian function in postpartum primiparous and pluriparous Mediterranean Italian buffaloes (Bubalus bubalis) during months of increasing daylength. Ovarian ultrasound monitoring was carried out for a total of 60 days from calving in 10 primiparous and 10 pluriparous buffaloes. Progesterone was determined from calving until a week after first postpartum ovulation. The study was undertaken during months of increasing day length. Time required for complete postpartum uterine involution was 31 +/- 1.0 and 33 +/- 1.3 days in primiparous and pluriparous buffaloes respectively (P = 0.1). The first postpartum ovulation was recorded on 4 primiparous and 8 pluriparous buffaloes (P = 0.16). Time for first postpartum ovulation to occur was 25.5 +/- 6.9 and 15.5 +/- 1.3 days in primiparous and pluriparous buffaloes, respectively (P = 0.07). Overall, 8 of the 12 first postpartum ovulations (66.6%) occurred in the ovary contra-lateral to the one bearing the gravidic CL, one out of 4 in primiparous and 3 out of 8 in pluriparous buffaloes (P = 1.0). Following a first postpartum ovulation, 3 primiparous and 4 pluriparous buffaloes displayed a complete wave of follicular development leading to a new ovulation. Ovulation following parturition was not recorded in 6 primiparous and two pluriparous buffaloes for the 60 days of ultrasound monitoring. Growth rate (mm/d) and largest size (mm) of first postpartum ovulating follicle was 0.95 +/- 0.18 and 1.07 +/- 0.07 (P = 0.4), and 13.5 +/- 0.8 and 14.1 +/- 0.4 (P = 0.4) in primiparous and pluriparous buffaloes, respectively. Following calving, the total number of available antral follicles (> or =2 mm) declined gradually towards the end of the study period. Follicles greater or equal to 3 mm in diameter on the contrary showed a prominent increase in the first 2 weeks from calving. The number of follicles greater or equal to 3 mm in diameter was significantly higher in the ovary contra-lateral to the one bearing the gravidic CL. A balance in the number of such follicles was reached toward the end of the first month. In conclusion, although some follicular activity was recorded in the ovaries of all buffaloes, true postpartum resumption of cyclicity in the months of increasing daylight hours was delayed in the majority of animals.     

Competitive reactions of a ruthenium arene anticancer complex with histidine, cytochrome c and an oligonucleotide

The ruthenium arene anticancer complex [(⁶-bip)Ru(en)Cl][PF₆] (1) (bip is biphenyl, en is ethylenediamine) reacted slowly with the amino acid L-histidine (L-His) in aqueous solution at 310 K. Two L-His adducts of 1 were separated by high-performance liquid chromatography and identified by electrospray ionisation mass spectrometry and NMR: an imidazole N-bound complex [(⁶-bip)Ru(en)(N–L-His)]²⁺, and an N-bound complex [(⁶-bip)Ru(en)(N–L-His)]²⁺. At 310 K, after 24 h only about 22% of complex 1 (2 mM) reacted with L-His, and of the unreacted 1, 59% had hydrolysed. In the presence of 100 mM NaCl, approximately 90% of 1 remained unreacted. In aqueous solution or triethylammonium acetate (TEAA) buffer (pH 7.6), ¹⁵N-labelled 1 reacted with cytochrome c to give two monoruthenated protein adducts. The reaction reached equilibrium within 2 h by which time approximately 50% of cytochrome c was ruthenated. On the basis of [¹H, ¹⁵N] NMR data, one adduct may have Ru bound to the N-terminus, and the other to a carboxylate group on the protein. In TEAA buffer and at 310 K, more than 90% of the 14-mer oligonucleotide d(TATGTACCATGTAT) reacted with 2 mol Eq of 1 to give rise to monoruthenated and diruthenated oligonucleotide adducts. The presence of cytochrome c (1 mol Eq) or L-His (4 mol Eq) had little effect on the course of the reaction with the oligonucleotide. In cells, DNA (or RNA) may be a favoured reaction site for this Ru anticancer complex.Electronic supplementary material is available for this article at .

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