Systematic enhancement of polymerization of recombinant sickle hemoglobin mutants: implications for transgenic mouse model for sickle cell anemia

To provide quantitative information on the sites that promote polymerization of sickle hemoglobin (HbS) after formation of the initial hydrophobic bond involving Val-6(beta) [E6V(beta)] and also to provide hemoglobins with an enhanced polymerization that could be used in a mouse model for sickle cell anemia, we have expressed recombinant double, triple, and quadruple HbS mutants with substitutions on both the alpha- and beta-chains, E6V(beta)/E121R(beta), D75Y(alpha)/E6V(beta)/E121R(beta) and D6A(alpha)/D75Y(alpha)/E6V(beta)/E121R(beta). These recombinant hemoglobins were extensively characterized by high-performance liquid chromatography analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, amino acid analysis, and mass spectroscopy. They retained the functional properties of the Hb tetramer and polymerized in a linear manner at progressively lower Hb concentration as a function of the degree of substitution, suggesting that these remote sites (alphaD6A, alphaD75Y, and betaE121R) on the alpha- and beta-chains exhibit additive, enhanced polymerization properties. The quadruple mutant has a polymerization concentration close to that of the purified SAD hemoglobin from transgenic mouse red blood cells consisting of HbS, Hb Antilles, and Hb D-Punjab. Normal mouse Hb increases the polymerization concentration of each mutant. Thus, the general approach of using recombinant Hbs as described here should prove useful in elucidating the quantitative aspects of the mechanism of HbS polymerization and in identifying the contribution of individual sites to the overall process. The strategy described here demonstrates the feasibility of a systematic approach to achieve future recombinant HbS mutants that could provide a new generation of the transgenic mouse model for sickle cell anemia.     

Deficiency of IL-5 receptor alpha-chain selectively influences the development of the common mucosal immune system independent IgA-producing B-1 cell in mucosa-associated tissues

Deletion of IL-5R alpha-chain (IL-5R alpha-/-) selectively influenced the mucosal IgA responses in vivo. While levels of IgA in mucosal secretions were more reduced in IL-5R alpha-/- mice than in wild-type mice, the levels of IgA in serum were not changed. The frequency of IgA-producing cells was reduced in mucosal effector sites (e.g., intestinal lamina propria and nasal passage), but not in inductive sites such as Payer's patches and nasal-associated lymphoreticular tissues in IL-5R alpha-/- mice. IgA-committed (surface IgA+; sIgA+) B-1 cells mainly resided in mucosal effector tissues, while conventional sIgA+ B (B-2) cells formed in mucosal inductive sites of wild-type mice. In contrast, in the effector tissue of IL-5R alpha-/- mice, sIgA+ B-1 cells, but not sIgA+ B-2 cells in the inductive site, were significantly reduced. IL-5R alpha was more expressed on sIgA+ B-1 cells than was IL-6R, while both IL-5R alpha and IL-6R were expressed on sIgA+ B-2 cells in wild-type mice. sIgA+ B-1 cells produced high levels of IgA with rIL-5 rather than of rIL-6 in vitro. Taken together, the findings suggest that the IL-5/IL-5R signaling pathway is critically important for the development of common mucosal immune system independent sIgA+ B-1 cell in mucosal effector tissues in vivo.     

Hemodynamic and inotropic effects of milrinone after heart transplantation in the setting of recipient pulmonary hypertension

The complementary fragments of human Hb alpha, alpha1-30, and alpha31-141 are spliced together by V8 protease in the presence of 30% n-propanol to generate the full-length molecule (Hb alpha-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb alpha is facilitated by the organic cosolvent induced alpha-helical conformation of product acting as the "molecular trap" of the splicing reaction. The segments alpha24-30 and alpha31-40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) alpha24-40 has been manipulated by engineering the amino acid replacements to the positions alpha27 and alpha31 to delineate the structural basis of the molecular trap. The location of Glu27 and Arg31 residues in the contiguous segment alpha24-40 (as well as in other larger segments) is ideal to generate (i, i + 4) side-chain carboxylate-guanidino interaction in its alpha-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at alpha27 and alpha31 facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The gamma-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an alpha-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu30-Arg31 peptide bond. The second structural aspect is a site-specific event, an i, i + 4 side-chain interaction in the alpha-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven "molecular traps" of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.     

Alpha-chain disease: analysis of alpha-chain protein and secretory component in jejunal fluid

BACKGROUND: It is unclear why different forms of alpha-chain disease protein appear in intestinal fluid. This was studied in a 23-year-old Mauritanian man in whom alpha-chain disease was diagnosed localized to the duodenum and jejunum, nasopharynx, and bone marrow. METHODS: The duodenal infiltrate was studied by immunohistochemistry. Forms of alpha chain-containing proteins in serum and jejunal fluid were analyzed by ultracentrifugation and radioimmunoassays. RESULTS: The infiltrating cells contained alpha-1 chain but no light chains, and approximately 66% showed variable expression of J chain. Serum contained a large fraction of monomeric alpha-chain disease protein, whereas both monomeric and heavier forms appeared in jejunal fluid. Some of the latter were bound to secretory component, and the fluid contained virtually no free component. CONCLUSIONS: Linkage of polymeric alpha-chain disease protein to secretory component depends on balanced synthesis of alpha chains and J chain in the proliferating B cells, giving rise to polymers with binding site for secretory component expressed as an epithelial receptor. Insufficient receptor-mediated transport capacity (either relative and/or because of intestinal crypt reduction) results in passive external transfer of polymers without bound secretory component along with leakage of serum-derived or locally produced monomeric alpha-chain disease protein, the latter presumably originating from immunocytes with little or no J-chain synthesis.     

Distinct roles for leukemia inhibitory factor receptor alpha-chain and gp130 in cell type-specific signal transduction

Leukemia inhibitory factor (LIF) induces a variety of disparate biological responses in different cell types. These responses are thought to be mediated through the functional LIF receptor (LIFR), consisting of a heterodimeric complex of LIFR alpha-chain (LIFRalpha) and gp130. The present study investigated the relative capacity of the cytoplasmic domains of each receptor subunit to signal particular responses in several cell types. To monitor the signaling potential of LIFRalpha and gp130 individually, we constructed chimeric receptors by linking the extracellular domain of granulocyte colony-stimulating factor receptor (GCSFR) to the transmembrane and cytoplasmic regions of either LIFRalpha or gp130. Both chimeric receptors and the full-length GCSFR in expressed in M1 myeloid leukemic cells to measure differentiation induction, in embryonic stem cells to measure differentiation inhibition, and in Ba/F3 cells to measure cell proliferation. Our results demonstrated that whereas GCSFR-gp130 receptor homodimer mediated a GCSF-induced signal in all three cell types, the GCSFR-LIFRalpha receptor homodimer was only functional in embryonic stem cells. These findings suggest that the signaling potential of gp130 and LIFRalpha cytoplasmic domains may differ depending upon the tissue and cellular response initiated.     

Mechanisms of macrophage stimulation through CD8: macrophage CD8alpha and CD8beta induce nitric oxide production and associated killing of the parasite Leishmania major

Prior studies demonstrated that rat macrophages express CD8, which differs from T lymphocyte CD8 within the ligand binding domain. We investigated whether stimulation of macrophage CD8 could induce mediator release and regulate host defense. Cross-linking either CD8alpha (OX8, 5 microg/ml) or CD8beta (341, 10 microg/ml) stimulated nitric oxide (NO) production, which correlated with an up-regulation of inducible NO synthase protein. Cell signaling inhibitors were used to elucidate the pathways of CD8alpha and CD8beta stimulation. Genistein (broad spectrum protein tyrosine kinase inhibitor, 10 microg/ml), PP1 (src family kinase inhibitor, 5 microg/ml), polymyxin B (protein kinase C (PKC) inhibitor, 100 microg/ml), and Ro 31-8220 (PKC inhibitor, 1 microM) significantly inhibited anti-CD8alpha- and anti-CD8beta-stimulated NO production and inducible NO synthase up-regulation, suggesting that tyrosine kinase(s) (src family) and PKC are involved in CD8 signaling. In addition, cross-linking CD8alpha stimulated NO-dependent macrophage killing of the parasite Leishmania major. For the first time, this work demonstrates that the beta-chain of macrophage CD8, in addition to the alpha-chain, can regulate mediator release. These results further illustrate the importance of this molecule and support our previous data demonstrating differences between macrophage and T lymphocyte CD8. Additional studies on the signaling mechanisms and possible ligand(s) for macrophage CD8 will lead to a greater understanding of inflammation and host defense.     

Haemoglobins of the shark, Heterodontus portusjacksoni II. Amino acid sequence of the alpha-chain

The amino acid sequence of the alpha-chain of the principal haemoglobin from the shark, H. portusjacksoni has been determined. The chain has 148 residues and is acetylated at the amino terminal. The soluble peptides obtained by tryptic and chymotryptic digestion of the protein or its cyanogen bromide fragments were isolated by gel filtration, paper ionophoresis and paper chromatography. The amino acid sequences were determined by the dansyl-Edman procedure. The insoluble "core" peptide from the tryptic digestion contained 34 residues and required cleavage by several prosteases before the sequence was established. Compared with human alpha-chain there are 88 amino acid differences including the additional seven residues which appear on the amino terminal of the shark chain. There is also one deletion and one insertion. The chain contains no tryptophan but has four cysteinyl residues which is the highest number of such residues recorded for a vertebrate globin. In the alpha1beta1 contact sites there are four changes in the oxyhaemoglobin form and six deoxy form. Nine of the 16, alpha1beta1 contact sites show variation while three of the haem contact sites have changed in comparison to the residues known to be involved in these interactions in horse haemoglobin alpha-chain. Use of the sequence data to estimate a time of divergence of the shark from the main vertebrate line yielded the value of 410 +/- 46 million years. The data, in general, support the palaeontological view that bony fishes arose before the elasmobranchs.     

Ligand-gated ion channel subunit partnerships: GABAA receptor alpha6 subunit gene inactivation inhibits delta subunit expression

Cerebellar granule cells express six GABAA receptor subunits abundantly (alpha1, alpha6, beta2, beta3, gamma2, and delta) and assemble various pentameric receptor subtypes with unknown subunit compositions; however, the rules guiding receptor subunit assembly are unclear. Here, removal of intact alpha6 protein from cerebellar granule cells allowed perturbations in other subunit levels to be studied. Exon 8 of the mouse alpha6 subunit gene was disrupted by homologous recombination. In alpha6 -/- granule cells, the delta subunit was selectively degraded as seen by immunoprecipitation, immunocytochemistry, and immunoblot analysis with delta subunit-specific antibodies. The delta subunit mRNA was present at wild-type levels in the mutant granule cells, indicating a post-translational loss of the delta subunit. These results provide genetic evidence for a specific association between the alpha6 and delta subunits. Because in alpha6 -/- neurons the remaining alpha1, beta2/3, and gamma2 subunits cannot rescue the delta subunit, certain potential subunit combinations may not be found in wild-type cells.     

Crystal structure of deoxy-human hemoglobin beta6 Glu --> Trp. Implications for the structure and formation of the sickle cell fiber

An atomic-level understanding of the interactions between hemoglobin molecules that contribute to the formation of pathological fibers in sickle cell disease remains elusive. By exploring crystal structures of mutant hemoglobins with altered polymerization properties, insight can be gained into sickle cell hemoglobin (HbS) polymerization. We present here the 2.0-A resolution deoxy crystal structure of human hemoglobin mutated to tryptophan at the beta6 position, the site of the glutamate --> valine mutation in HbS. Unlike leucine and isoleucine, which promote polymerization relative to HbS, tryptophan inhibits polymerization. Our results provide explanations for the altered polymerization properties and reveal a fundamentally different double strand that may provide a model for interactions within a fiber and/or interactions leading to heterogeneous nucleation.     

Lactate, lactate/pyruvate ratio and catecholamine interrelations in cord blood at delivery in complicated pregnancies

Fucosyltransferases are the enzymes transferring fucose from GDP-Fuc to Gal in an alpha1,2-linkage and to GlcNAc in alpha1,3-, alpha1,4-, or alpha1,6-linkages. Since all fucosyltransferases utilize the same nucleotide sugar, their specificity will probably reside in the recognition of the acceptor and in the type of linkage formed. A search of nucleotide and protein databases yielded more than 30 sequences of fucosyltransferases originating from mammals, chicken, nematode, and bacteria. On the basis of protein sequence similarities, these enzymes can be classified into four distinct families: (1) the alpha-2-fucosyltransferases, (2) the alpha-3-fucosyltransferases, (3) the mammalian alpha-6-fucosyltransferases, and (4) the bacterial alpha-6-fucosyltransferases. Nevertheless, using the sensitive hydrophobic cluster analysis (HCA) method, conserved structural features as well as a consensus peptide motif have been clearly identified in the catalytic domains of all alpha-2 and alpha-6-fucosyltranferases, from prokaryotic and eukaryotic origin, that allowed the grouping of these enzymes into one superfamily. In addition, a few amino acids were found strictly conserved in this family, and two of these residues have been reported to be essential for enzyme activity for a human alpha-2-fucosyltransferase. The alpha-3-fucosyltransferases constitute a distinct family as they lack the consensus peptide, but some regions display similarities with the alpha-2 and alpha-6-fucosyltranferases. All these observations strongly suggest that the fucosyltransferases share some common structural and catalytic features.     

Inhibitory effect of magnosalin derived from Flos magnoliae on tube formation of rat vascular endothelial cells during the angiogenic process

An aqueous water extract of Flos magnoliae, a Japanese Sino-medicine, inhibits angiogenesis in adjuvant-induced mouse pouch granuloma. Magnosalin (MSA) and magnoshinin (MSI), neolignans isolated from magnolia, have a crucial role in the anti-angiogenic effect of magnolia (Kimura et al., Int. Arch. Allergy Appl. Immunol., 93, 365 (1990); Phytother. Res., 6, 209 (1992)). We investigated the effects of these neolignans on tube formation of endothelial cells (EC) cultured in type I collagen gel during the angiogenic process. MSA (0.1-10 microM), MSI (0.23-7 microM) and corticosterone (CS: 0.3-30 microM) inhibited fetal bovine serum (FBS)-stimulated tube formation in a concentration-dependent manner. Their 30% inhibitory concentration (IC30, 95% confidence limits) values were 0.51 (0.20-1.27) for MSA, 8.14 (2.48-26.7) for MSI and 3.65 microM (2.47-5.40) for CS, respectively. MSA and MSI (1-3 microM) also inhibited interleukin (IL)-1 alpha-stimulated tube formation in a concentration-dependent manner. Their IC50 values (95% confidence limits) were 1.22 (1.01-1.47) for MSA and 0.74 microM (0.24-2.31) for MSI against a submaximal concentration (69 pM) of IL-1 alpha-stimulated tube formation. Their inhibitory effects on the action of IL-1 alpha were non-competitive. These results demonstrate that MSA inhibited FBS-stimulated tube formation with a greater potency than MSI. The inhibitory effect of MSA on the action of FBS differed from that on the action of IL-1 alpha.     

Alpha-1A adrenergic receptor stimulation with phenylephrine promotes arachidonic acid release by activation of phospholipase D in rat-1 fibroblasts: inhibition by protein kinase A

This study was conducted to determine the mechanism of arachidonic acid (AA) release elicited by phenylephrine (PHE) stimulation of alpha adrenergic receptor (AR), and its modulation by cyclic adenosine 3',5'-monophosphate (cAMP) in Rat-1 fibroblasts (R-1Fs) transfected with the alpha-1A, alpha-1B or alpha-1D AR. PHE increased AA release and also caused a marked accumulation of cAMP in R-1Fs expressing the alpha-1 AR subtypes, but not in those transfected with vector alone. PHE also enhanced phospholipase D (PLD), but not phospholipase A2 (PLA2) activity. The increase in PHE-induced AA release, PLD activity and cAMP accumulation differed among the various alpha AR subtypes with: alpha-1A > alpha-1B > alpha-1D AR. The effect of PHE to increase AA release was attenuated by C2-ceramide, an inhibitor of PLD; propranolol, a phosphatidate phosphohydrolase inhibitor; and RHC-80267, a diacylglycerol lipase inhibitor in R-1Fs expressing the alpha-1A AR. Forskolin, which activates adenylyl cyclase, increased cAMP accumulation and inhibited PHE-induced AA release and PLD activity in alpha-1A-AR-expressing R-1Fs. 8-(4-chlorophenyl-thio)-cAMP, a nonhydrolyzable analog of cAMP, also attenuated the rise in AA release and PLD activity elicited by PHE in these cells. In contrast, SQ 22536, an adenylyl cyclase inhibitor, and KT 5720, a protein kinase A inhibitor, increased PHE-induced AA release and PLD activity in R-1Fs expressing the alpha-1A AR. These data suggest that the alpha-1A, alpha-1B and alpha-1D ARs are coupled to PLD activation and cAMP accumulation. Moreover, PHE promotes AA release in R-1Fs expressing the alpha-1A AR through PLD activation. Furthermore, cAMP generated by alpha-1A AR stimulation acts as an inhibitory modulator of PLD activity and AA release via protein kinase A.     

The occurrence and identification of alpha-thalassemia-2 among hemoglobin S heterozygotes

The in-vitro synthesis of hemoglobin (Hb) chains was studied among 60 Hb S heterozygotes (AS) having different quantities of Hb S, including five with an associated alpha-chain heterozygosity (ASAG). Hematologic values and hemoglobin composition were studied in these cases and in 15 other ASAG heterozygotes. The percentages of Hb S (which fell between 27% and 42%) and the mean corpuscular volume values correlated directly with the alpha/non-alpha values, confirming previous suggestions (Huisman, Hemoglobin 1:349, 1977) that the concomitant occurrence of an alpha-thalassemia-2 heterozygosity (alpha alpha(0)/alpha alpha; beta/beta(S)) or homozygosity (alpha(0) alpha/alpha(0) alpha; beta/beta(S)) resulted in intermediate or lower levels of Hb S compared with Hb S heterozygotes having four active alpha-chain genes (alpha alpha/alpha alpha; beta/beta(S)). Among ASAG heterozygotes, the occurrence of low (about 25%), intermediate (about 33%), or high (about 45%) proportions of an alpha-chain variant resulting from a variability in the number of active alpha-chain genes due to alpha-thal-2 coincided with high (39%), intermediate (34%), or low (28%) levels of Hb S, respectively. However, the overlap of biosynthetic data between Hb S heterozygotes with four, three, or two active alpha-chain genes prevents a reliable diagnosis in individual cases.     

1alpha,25-dehydroxyvitamin D3 synergism toward transforming growth factor-beta1-induced AP-1 transcriptional activity in mouse osteoblastic cells via its nuclear receptor

The present study demonstrates 1alpha,25-dehydroxyvitamin D3 (1alpha-25-(OH)2D3) synergism toward transforming growth factor (TGF)-beta1-induced activation protein-1 (AP-1) activity in mouse osteoblastic MC3T3-E1 cells via the nuclear receptor of the vitamin. 1alpha-25-(OH)2D3 synergistically stimulated TGF-beta1-induced expression of the c-jun gene in the cells but not that of the c-fos gene. We actually showed by a gel mobility shift assay 1alpha-25-(OH)2D3 synergism of TGF-beta1-induced AP-1 binding to the 12-(O-tetradecanoylphorbol-13-acetate response element (TRE). 1alpha-25-(OH)2D3 markedly stimulated the transient activity of TGF-beta1-induced AP-1 in the cells transfected with a TRE-chloramphenicol acetyltransferase (CAT) reporter gene. Also, a synergistic increase in TGF-beta1-induced CAT activity was observed in the cells cotransfected with an expression vector encoding vitamin D3 receptor (VDR) and the reporter gene. However, the synergistic CAT activity was inhibited by pretreatment with VDR antisense oligonucleotides. In addition, in a Northern blot assay, we observed 1alpha-25-(OH)2D3 synergism of TGF-beta1-induced expression of the c-jun gene in the cells transfected with the VDR expression vector and also found that the synergistic action was clearly blocked by VDR antisense oligonucleotide pretreatment. The present study strongly suggests a novel positive regulation by 1alpha-25-(OH)2D3 of TGF-beta1-induced AP-1 activity in osteoblasts via "genomic action."     

The formation or the reduction of a disulfide bridge on the gamma subunit of chloroplast ATP synthase affects the inhibitory effect of the epsilon subunit

We have studied the change of the catalytic activity of chimeric complexes that were formed by chloroplast coupling factor 1 (CF1) -gamma, alpha and beta subunits of thermophilic bacterial F1 after formation or reduction of the disulfide bridge of different gamma subunits modified by oligonucleotide-directed mutagenesis techniques. For this purpose, three mutant gamma subunits were produced: gamma Delta194-230, here 37 amino acids from Pro-194 to Ile-230 are deleted, gammaC199A, Cys-199 is changed to Ala, and gamma Delta200-204, amino acids from Asp-200 to Lys-204 are deleted. All of the chimeric subunit complexes produced from each of these mutant CF1-gamma subunits and alpha and beta subunits from thermophilic bacterial F1 lost the sensitivity against thiol reagents when compared with the complex containing wild-type CF1-gamma. The pH optimum (pH 8.5-9.0) and the concentration of methanol to stimulate ATPase activities were not affected by these mutations. These indicate that the introduction of the mutations did not change the main features of ATPase activity of the chimeric complex. However, the interaction between gamma subunit and epsilon subunit was strongly influenced by the type of gamma subunit itself. Although the ATPase activity of the chimeric complex that contained gamma Delta200-204 or gammaC199A was inhibited by the addition of recombinant epsilon subunit from CF1 similarly to complexes containing the reduced wild-type gamma subunit, the recombinant epsilon subunit did not inhibit the ATPase of the complex, which contained the oxidized form of gamma subunit. Therefore the affinity of the epsilon subunit to the gamma subunit may be dependent on the state of the gamma subunit or the epsilon subunit may bind to the oxidized form of gamma subunit in a mode that does not inhibit the activity. The ATPase activity of the complex that contains gamma Delta194-230 was not efficiently inhibited by epsilon subunit. These results show that the formation or reduction of the disulfide bond on the gamma subunit may induce a conformational change in the region that directly affects the interaction of this subunit with the adjacent epsilon subunit.     

Characterization of visually similar diffuse diseases from B-scan liver images using nonseparable wavelet transform

BACKGROUND/AIMS: The serine proteinase inhibitors (serpins), alpha-1-antitrypsin (AAT) and alpha-1-antichymotrypsin (ACT), are acute phase proteins synthesized by hepatocytes and excreted to some extent into bile. Their role in gallstone pathogenesis is unclear, and it was the aim of this study to determine their effect on cholesterol crystal growth rate in model bile. METHODS: Purified AAT and ACT were added to model bile at concentrations from 0.5 to 500 microg/ml. Cholesterol crystal growth was analyzed daily by polarizing microscopy and spectrophotometrically at 650 nm. Serpin inhibitory activity was measured spectrophotometrically at 405 nm, and polymerization was studied on 7.5% SDS-PAGE under non-reducing conditions, by immunoelectrophoresis and Western blotting. RESULTS: ACT added to model bile at a concentration of 0.5 microg/ml, inhibited cholesterol crystallization by 30%, had no influence at 5 microg/ml, and increased the crystallization rate 2-3 fold at concentrations of 50 and 500 microg/ml. AAT at a concentration of 0.5 microg/ml had a profound (50%) inhibitory effect on cholesterol crystal growth rate, lacked significant effect at both concentrations of 5 and 50 microg/ml, and showed stimulation of crystal growth up to 30% at a concentration of 500 microg/ml. Both serpins incubated in model bile polymerized and totally lost their inhibitory activity. CONCLUSIONS: Serpins can exhibit both inhibiting and promoting effects on the cholesterol crystallization rate in model bile, dependent on their concentrations. Since AAT and ACT are acute phase proteins, their concentrations may vary under certain pathological conditions, which may result in different actions of these serpins in gallstone formation.     

Synthesis and in vitro activity of some epimeric 20 alpha-hydroxy, 20-oxime and aziridine pregnene derivatives as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase and 5 alpha-reductase

Some epimeric 20-hydroxy, 20-oxime, 16 alpha, 17 alpha-, 17,20- and 20,21-aziridine derivatives of progesterone were synthesized and evaluated as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase (P450(17) alpha) and 5 alpha-reductase (5 alpha-R). The reduction of 16-dehydropregenolone acetate (3a) was reinvestigated. NaBH4 in the presence of CeCl3 gave better stereo-selectivity for 20 beta-ol [20 alpha/20 beta-OH (4 alpha/4 beta) = 1/2.7] than LTBAH or the Meerwein-Pondroff method reported; reduction with Zn in HOAc formed exclusively 20 alpha-ol (4 alpha b). The 20 alpha- and 20 beta-hydroxy-4,16-pregnadien-3-one (9 alpha) and (9 beta) were synthesized from the alcohols 4 alpha b and 4 beta b. Several 20-oxime pregnadienes and 16 alpha, 17 alpha-, 17,20- and 20,21-aziridinyl-5-pregnene derivatives were also synthesized. LiAlH4 reduction of the 16-en-20-oxime (12b) yielded 20 (R)-(13a) and 20(S)-17 alpha,20-aziridine (13b) and 20(R)-17 beta,20-aziridine (14a). Several compounds inhibited the human P450(17) alpha with greater potency than ketoconzole. The 5 alpha-R enzyme assay showed that while (9 alpha) did not have any activity, (9 beta) and (3b) were potent 5 alpha-reductase (IC50 = 21 and 31 nM) inhibitors with activities similar to finasteride. The 20-oximes (17a) and (17b) were potent dual inhibitors for both 5 alpha-R (IC50 = 63 and 115 nM, compared to 33 nM for finasteride) and P450(17) alpha (IC50 = 43 and 25 nM, compared to 78 nM for ketoconazole).     

Pure amnesia after unilateral left polar thalamic infarct: topographic and sequential neuropsychological and metabolic (PET) correlations

The present study examined the effect of sodium salicylate and indomethacin on the recruitment of osteoclast-like cells in vitro. When mouse bone marrow cells were cultured for 8 days with 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3, 10(-8) M), prostaglandin E2 (PGE2, 10(-6) M) and recombinant human interleukin-1 alpha (rHIL-1 alpha, 2 ng/ml), numerous tartrate-resistant acid phosphatase (TRAP-positive) multinucleated cells (MNCs) formed. Adding sodium salicylate or indomethacin inhibited the formation of TRAP-positive MNCs in a dose-dependent manner. This inhibitory effect was more pronounced when the drugs were given at a later stage in the culture period. Indomethacin appeared to be more potent than sodium salicylate. PGE2 production was inhibited by sodium salicylate or indomethacin. Exogenous PGE2 failed to overcome the inhibitory effect of both drugs. These results suggest that sodium salicylate and indomethacin have inhibitory effects on the recruitment of osteoclast-like MNCs, preferentially on the later stage, and that PGE2 is not the only compound targeted by these drugs in reducing osteoclast-like cell formation in mouse bone marrow culture.