Solvent-Controlled, Tunable Domino Reaction of 3-Ylideneoxindoles with in Situ-Generated α-Aryldiazomethanes: A Facile Access to 3-Spirocyclopropyl-2-oxindole and Pyrazoloquinazolinone Scaffolds.

A mild and efficient solvent-controlled, metal-free switchable 1,3-dipolar cycloaddition/ring contraction or ring expansion domino reaction of 3-ylideneoxindoles with in situ-generated α-aryldiazomethanes has been developed. This domino reaction provided a series of aryl-substituted 3-spirocyclopropyl-2-oxindoles and pyrazoloquinazolinones with excellent regio- and diastereoselectivity from common substrates under varying solvent conditions.

Synthesis, DNA binding affinity and anticancer activity of novel 4H-benzo[g][1,2,3]triazolo[5,1-c][1,4]oxazocines.

A new class of tricyclic heterocycles 4H-benzo[g][1,2,3]triazolo[5,1-c][1,4]oxazocines was synthesized through a Knoevenagel condensation/azide-alkyne cycloaddition reaction cascade in one-pot operation. These eight membered ring containing heterocycles exhibited moderately high anticancer activity against four cancer cell lines; human cervix cancer cell line (HeLa), human prostate cancer cell line (DU145), human breast cancer cell line (MCF-7) and human breast adenocarcinoma epithelial cell line (MDA-MB-231). Our results indicate that these compounds have a weak cytotoxic effect on normal human mammary epithelial cell line (MCF-10A). Cell cycle and apoptosis assay indicate that they inhibit the cell cycle at the G2/M phase and induce apoptosis. Through the RED100 assay, it is evident that they have potential to inhibit pBR 322 plasmid DNA cleavage by BamH1. UV-visible, fluorescence titration and viscosity studies suggested that these compounds possess DNA binding affinity.

One-pot, three-component approach to the synthesis of 3,4,5-trisubstituted pyrazoles.

An operationally simple and high yielding protocol for the synthesis of polyfunctional pyrazoles has been developed through one-pot, three-component coupling of aldehydes, 1,3-dicarbonyls, and diazo compounds as well as tosyl hydrazones. The reaction proceeds through a tandem Knoevenagel condensation, 1,3-dipolar cycloaddition, and transition metal-free oxidative aromatization reaction sequence utilizing molecular oxygen as a green oxidant. The scope of the reaction was studied by varying the aldehyde, 1,3-dicarbonyl, and diazo component individually.

Effect of ciprofloxacin on specific immune response in rabbits.

Ciprofloxacin (10 mg/kg body weight, iv, twice daily for 4 days) failed to alter specific antibody titres, total immunoglobulin concentration, total serum protein concentration, total leukocyte count, lymphocyte percentage, phagocytic index and skin thickness in DNCB skin sensitivity test against Brucella plain killed antigen in New Zealand White rabbits. It can be concluded that ciprofloxacin at the dose and duration employed did not adversely affect specific immune response in normal rabbits.

The human gC1qR/p32 gene, C1qBP. Genomic organization and promoter analysis.

gC1qR is an ubiquitously expressed cell protein that interacts with the globular heads of C1q (gC1q) and many other ligands. In this study, the 7.8-kilobase pair (kb) human gC1qR/p32 (C1qBP) gene was cloned and found to consist of 6 exons and 5 introns. Analysis of a 1.3-kb DNA fragment at the 5'-flanking region of this gene revealed the presence of multiple TATA, CCAAT, and Sp1 binding sites. Luciferase reporter assays performed in different human cell lines demonstrated that the reporter gene was ubiquitously driven by this 1.3-kb fragment. Subsequent 5' and 3' deletion of this fragment confined promoter elements to within 400 base pairs (bp) upstream of the translational start site. Because the removal of the 8-bp consensus TATATATA at -399 to -406 and CCAAT at -410 to -414 did not significantly affect the transcription efficiency of the promoter, GC-rich sequences between this TATA box and the translation start site may be very important for the promoter activity of the C1qBP gene. One of seven GC-rich sequences in this region binds specifically to PANC-1 nuclear extracts, and the transcription factor Sp1 was shown to bind to this GC-rich sequence by the supershift assay. Primer extension analysis mapped three major transcription start regions. The farthest transcription start site is 49 bp upstream of the ATG translation initiation codon and is in close proximity of the specific SP1 binding site.

Enhanced anti-influenza activity of a surfactant protein D and serum conglutinin fusion protein.

We previously demonstrated that bovine serum conglutinin has markedly greater ability to inhibit influenza A virus (IAV) infectivity than other collectins. We now show that recombinant conglutinin and a chimeric protein containing the NH(2) terminus and collagen domain of rat pulmonary surfactant protein D (rSP-D) fused to the neck region and carbohydrate recognition domain (CRD) of conglutinin (termed SP-D/Cong(neck+CRD)) have markedly greater ability to inhibit infectivity of IAV than wild-type recombinant rSP-D, confirming that the potent IAV-neutralizing activity of conglutinin resides in its neck region and CRD. Furthermore, by virtue of incorporation of the NH(2) terminus and collagen domain of SP-D, SP-D/Cong(neck+CRD) caused substantially greater aggregation of IAV particles and enhancement of neutrophil binding of, and H(2)O(2) responses to, IAV than recombinant conglutinin or recombinant rSP-D. Hence, SP-D/Cong(neck+CRD) combined favorable antiviral and opsonic properties of conglutinin and SP-D. This study demonstrates an association of specific structural domains of SP-D and conglutinin with specific functional properties and illustrates that antimicrobial activities of wild-type collectins can be enhanced through recombinant strategies.

Pulmonary surfactant proteins A and D enhance neutrophil uptake of bacteria.

The collectins are a class of collagenous lectin proteins present in serum and pulmonary secretions [pulmonary surfactant protein (SP) A and SP-D] that are believed to participate in innate immune responses to various pathogens. With the use of flow cytometric and fluorescent-microscopic assays, SP-A and SP-D were shown to increase calcium-dependent neutrophil uptake of Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus. Evidence is provided that the collectins enhanced bacterial uptake through a mechanism that involved both bacterial aggregation and direct actions on neutrophils. The degree of multimerization of SP-D preparations was a critical determinant of both aggregating activity and potency in enhancing bacterial uptake. The mechanisms of opsonizing activity of SP-D and SP-A differed in important respects from those of opsonizing antibodies. These results provide the first evidence that surfactant collectins may promote neutrophil-mediated clearance of bacteria in the lung independently of opsonizing antibody.

The systemic lupus erythematosus (SLE) disease autoantigen-calreticulin can inhibit C1q association with immune complexes.

Following its release from cells during infection and inflammation, calreticulin (CRT) can act as an autoantigen in diseases such as SLE. Why CRT is a target of protective immunity and whether it may interfere with innate immunity once released from cells during inflammation is unclear. In the present study, we found that CRT was detected more frequently in SLE sera and in higher amounts than found in control sera. Approximately 40% of SLE sera tested contained autoantibodies against CRT as detected by ELISA and immunoblotting. CRT was found to be predominantly in the sera of SLE patients associated with immune complexes and C1q, and only bound to the surfaces of neutrophils in the presence of low levels of calcium and magnesium. In order to further investigate the C1q-CRT interaction, recombinant CRT and its discrete domains (N-, P-, and C-domains) were produced in Escherichia coli. CRT binds to globular head region of C1q primarily via its N- and P-domains. The N-domain was shown to be the most autoantigenic region of CRT, as the anti-CRT autoantibodies from most patients reacted against this region. CRT also altered C1q-mediated immune functions. The P-domain of CRT bound to C1q and reduced the binding of immune complexes in SLE sera to immobilized C1q. Full length CRT and its N- and P-domains were able to reduce the C1q-dependent binding of immune complexes to neutrophils and solid-phase bound C1q. We conclude that CRT, once released from leucocytes during inflammation, may not only induce an antigenic reaction, but also interfere with C1q-mediated inflammatory processes.

Release of calreticulin from neutrophils may alter C1q-mediated immune functions.

Calreticulin is an abundant intracellular protein which is involved in a number of cellular functions. During cytomegalovirus infection, as well as inflammatory episodes in autoimmune disease, calreticulin can be released from cells and detected in the circulation, where it may act as an immunodominant autoantigen in diseases such as systemic lupus erythematosus. Calreticulin is known to bind to the molecules of innate immunity, such as C1q, the first subcomponent of complement. However, the functional implications of C1q-calreticulin interactions are unknown. In the present study we sought to investigate, in greater detail, the interaction between these two proteins following the release of calreticulin from neutrophils upon stimulation. In order to pinpoint the regions of interaction, recombinant calreticulin and its discrete domains (N-, P- and C-domains) were produced in Escherichia coli. Both the N- and P-domains of calreticulin were shown to bind to the globular head regions of C1q. Calreticulin also appeared to alter C1q-mediated immune functions. Binding of calreticulin to C1q inhibited haemolysis of IgM-sensitized erythrocytes. Both the N- and P-domains of calreticulin were found to contain sites involved in the inhibition of C1q-induced haemolysis. Full-length calreticulin, and its N- and P-domains, were also able to reduce the C1q-dependent binding of immune complexes to neutrophils. We conclude that calreticulin, once released from neutrophils during inflammation, may not only induce an antigenic reaction, but, under defined conditions, may also interfere with C1q-mediated inflammatory processes.

Characterization of two mannose-binding protein cDNAs from rhesus monkey (Macaca mulatta): structure and evolutionary implications.

Mannose-binding proteins (MBPs), members of the collectin family, have been implicated as lectin opsonins for various viruses and bacteria. Two distinct but related MBPs, MBP-A and MBP-C, with approximately 55% identity at the amino acid level, have been previously characterized from rodents. In humans, however, only one form of MBP has been characterized. In this paper we report studies elucidating the evolution of primate MBPs. ELISA and Western blot analyses indicated that rhesus and cynomolgus monkeys have two forms of MBP in their sera, while chimpanzees have only one form, similar to humans. Two distinct MBP cDNA clones were isolated and characterized from a rhesus monkey liver cDNA library. Rhesus MBP-A is closely related to the mouse and rat MBP-A, showing 77% and 75% identity at the amino acid level, respectively. Rhesus MBP-A also has three cysteines at the N-terminus, similar to mouse and rat MBP-A and human MBP. Rhesus MBP-C shares 90% identity with the human MBP at the amino acid level and has three cysteines at the N-terminus, in contrast to two cysteine residues found in rodent MBP-C. A stretch of nine amino acids close to the N-terminus, absent in both mouse and rat MBP-A, but present in rodent MBP-C, chicken and human MBPs, is also found in the rhesus MBP-A. The phylogenetic analysis of rhesus and other mammalian MBPs, coupled with the serological data suggest that at least two distinct MBP genes existed prior to mammalian radiation and the hominoid ancestor apparently lost one of these genes or failed to express it.

Mouse surfactant protein-D. cDNA cloning, characterization, and gene localization to chromosome 14.

Surfactant protein-D (SP-D) is a collectin found associated with surfactant in the lung. SP-D has also been functionally characterized as an opsonin for diverse microorganisms and a chemoattractant for phagocytic cells. To determine the structure of mouse SP-D, we isolated and characterized clones from a B6/CBAF1J strain lung cDNA library using a PCR-derived genomic probe. The deduced sequence predicts a 19-amino acid signal sequence, a 25-amino acid long NH2 terminus with two cysteines, followed by an uninterrupted collagen domain with 59 Gly-X-Y repeats. Next, a short "neck" domain of 28 amino acids, with a potential to form trimeric alpha-helical coiled coil is found ending in a COOH-terminal 125-amino acid carbohydrate recognition domain. The mature mouse SP-D protein of 355 amino acids shows strong homology to rat (92% identity), human (76%), and bovine (72%) SP-D amino acid sequences. Northern blot and RT-PCR analysis revealed that the mouse SP-D gene is expressed predominantly in lung and, surprisingly, also in heart, stomach, and kidney but not in brain. In contrast, mouse surfactant protein-A (SP-A) mRNA expression was found to be restricted to lung. Human lung and stomach, but not heart or liver, were found to express SP-D mRNA, as determined by PCR. The mouse SP-D gene (Sftp4) has been localized to chromosome 14 (to a region syntenic to human chromosome 10), closely linked to the genes for other collagenous lectins, mannose-binding protein-A (MbI1), and SP-A (Sftp1).

Identification of a gC1q-binding protein (gC1q-R) on the surface of human neutrophils. Subcellular localization and binding properties in comparison with the cC1q-R.

Human neutrophils have multiple C1q-binding proteins. Direct ligand-binding studies with the globular domain of C1q and two-dimensional Western blot analysis revealed two gC1q-binding proteins (gC1q-R): a 33,000 M(r) protein (pI 4.5) mainly in the neutrophil plasma membrane and an 80,000-90,000 M(r) protein (pI 4.1-4.2) located mainly in the granules. Direct binding studies showed that C1q bound to this higher molecular weight protein under physiological conditions. In contrast, anti-cC1q-R antibody, which recognizes a protein binding to collagenous tails of C1q, detected only a 68,000 M(r) protein in the plasma membrane. Both the 33,000 and 68,000 M(r) receptors appear early on the surface of differentiating HL-60 cells. On mature neutrophils, surface expression of both C1q receptors was evident, but no upregulation was observed upon stimulation. Phorbol myristate acetate treatment of neutrophils downregulated both the receptors from cell surface, and significant amounts of soluble gC1q-R were in cell media supernatants, suggesting receptor shedding or secretion. gC1q-R, unlike cC1q-R, did not bind to other C1q-like ligands, namely mannose binding protein, surfactant protein-A, surfactant protein-D, or conglutinin under normal ionic conditions, suggesting a greater specificity for C1q than the "collectin" type receptor (cC1q-R). Rather, gC1q-R only bound purified C1q, and the binding was enhanced under low ionic conditions and in the absence of calcium. The role of C1q receptor shedding and its biologic consequence remain to be defined, but may contribute to the diversity of C1q-mediated responses observed in many cell types.

Characterization of murine mannose-binding protein genes Mbl1 and Mbl2 reveals features common to other collectin genes.

Mannose-binding protein (MBP) is a member of a family of collagenous lectins (collectins), which are believed to play an important role in first-line host defense. In this study, the two genes encoding MBP in mice--Mbl1 and Mbl2--have been isolated and their exon-intron structure studied to understand their evolutionary relationship to the single human (MBL) and the two rat MBP genes. Mouse Mbl1 and Mbl2 have five and six exons, respectively. The structure of the mouse Mbl genes is similar to that of the rat and human MBP genes and shows homology to the other collectin genes, with the entire carbohydrate recognition domain being encoded in a single exon and all introns being in phase 1. The MBP encoded by mouse Mbl1 with three cysteines in the first coding exon, like the rat Mbl1 and human MBL, is capable of a higher degree of multimerization and has apparent ability to fix complement in the absence of antibody or C1q. However, the structural features of other exons, that is, the larger size of collagen domain region in the first coding exon (64 bp in Mbl2 vs 46 bp in Mbl1) and the smaller size of the exon encoding the trimerization domain (69 bp in Mbl2 vs 75 bp in Mbl1) reveal that the single human MBL gene is closely related to rodent Mbl2 rather than rodent Mbl1. The findings in this study suggest that in contrast to the evolution of another collectin gene--bovine surfactant protein-D--which duplicated in bovidae after divergence from humans, MBP gene most likely duplicated prior to human-rodent divergence, and that the human homolog to Mbl1 was perhaps lost during evolution.

Characterization of the human neutrophil C1q receptor and functional effects of free ligand on activated neutrophils.

The partial characterization and expression of the C1q receptor (C1q-R) in relation to other complement receptors present on the surface of neutrophils has been examined, as well as the effects of free C1q on cell function. A polyclonal anti-C1q-R antibody recognizes a 68-kD neutrophil surface protein. C1q-R expression was not upregulated upon warming, priming, or exposure to FMLP, but decreased after exposure to phorbol myristate acetate (PMA), because of shedding of the receptor into the extracellular medium, as detected by enzyme-linked immunosorbent assay. CR3 and CR1 expression was upregulated from intracellular pools after cell stimulation by PMA. No evidence of intracellular pools of C1q-R was found, as assessed by immunoblotting of subcellular fractions. But C1q-R appeared to be expressed early in cell differentiation, was detected on undifferentiated HL-60 cells, and like CR3 expression, increased upon 5 days differentiation towards a neutrophil lineage. However, C1q-R expression decreased upon additional culture, whereas CR3 expression continued to increase. A large variation in the percentage of peripheral cells expressing C1q receptors in donors was observed, ranging from 13% to 100%, contrasting with CR3 receptors that exhibited less variability. Interactions between free monomeric C1q and neutrophils were also studied. Incubation of stimulated neutrophils with 10 to 100 micrograms/mL C1q resulted in a further increase in CR3 expression and adherence to albumin-coated surfaces. Staphylococci opsonized with low quantities of C1q (0.1 to 1 microgram/mL) mediated a moderate and sustained respiratory burst in neutrophils, whereas a burst of similar magnitude was generated only with free C1q at concentrations 10- to 100-fold higher. Stimulation was only partially inhibited if cells were first treated with anti-C1q-R antibody, suggesting other C1q binding proteins may be present on the cell surface. In summary, neutrophil C1q receptor is approximately 68-kD, exhibits varying expression on different subjects, and is not upregulated from intracellular stores on exposure to soluble stimuli. Stimulated, but not resting, neutrophils selectively respond to raised levels of free C1q, resulting in altered cell function and enhanced CR3 receptor expression. These studies thus suggest complex roles for C1q in neutrophil function.

Crystallization and preliminary X-ray analysis of a trimeric form of human mannose binding protein.

A trimeric form of the carbohydrate recognition domain of human mannose binding protein has been crystallized in two different forms. The first form crystallizes with symmetry consistent with space group P2(1)2(1)2(1) and a = 61 A; b = 144 A; c = 107 A with presumably two trimers in the asymmetric unit. The second form crystallizes with symmetry consistent with space group P321 and a = b = 77 A; c = 58 A and one monomer per asymmetric unit. The molecular and crystallographic 3-folds must be coincident in this crystal form.

Bovine conglutinin gene exon structure reveals its evolutionary relationship to surfactant protein-D.

Bovine conglutinin (BC), a member of the mammalian C-type collectin subfamily, is a serum protein synthesized in liver that is believed to play a role in natural host defense. Previously, we have characterized a full length BC cDNA and we now describe the partial characterization of a genomic clone that encodes for the BC gene (CGN1). BC is encoded by nine exons spanning > 11 kb and has been localized previously to band 18 of bovine (Bos taurus) chromosome 28. Genomic sequencing demonstrated that the signal peptide/amino-terminal domain, the carbohydrate recognition domain, and the linking peptide, a domain between the collagenous region and the carbohydrate recognition domain, are each encoded by a single exon. The collagenous domain is split into five exons, with the 5' most region being located within the exon that also encodes the signal peptide/amino terminus. The remaining four collagenous domain exons are tandemly arranged with lengths of 117, 108, 108, and 117 bp, respectively. Overall, the BC genomic organization is very similar to that of the human surfactant protein-D gene, SFTP4. On the basis of identical collagen domain structures, we suggest that conglutinin and bovine surfactant protein-D evolved from a gene duplication event occurring in Bovidae after divergence from other mammals.

Bovine conglutinin (BC) mRNA expressed in liver: cloning and characterization of the BC cDNA reveals strong homology to surfactant protein-D.

Bovine conglutinin (BC) is a C-type lectin isolated from bovine serum that appears to play a role in first-line host defense. The BC cDNA was cloned from a bovine liver library and the nucleotide (nt) sequence of 1519 bp was determined. The longest open reading frame encoded a 20-amino-acid (aa) signal sequence and a mature protein of 351 aa. Analysis of the nt and deduced aa sequences revealed 87 and 78% identity, respectively, with the sequences of another vertebrate lectin: bovine surfactant protein-D (SP-D). Of interest, the expression of the BC mRNA, as determined by RNase protection assay, is restricted to liver, unlike bovine SP-D, a lung-surfactant protein.