Physicochemical and biological properties of 6(1),6(3),6(5)-tri-O-alpha-maltosyl-cyclomaltoheptaose (6(1),6(3),6(5)-tri-O-alpha-maltosyl-beta-cyclodextrin)

A unique multibranched cyclomaltooligosaccharide (cyclodextrin, CD) of 6(1),6(3),6(5)-tri-O-alpha-maltosyl-cyclomaltoheptaose [6(1),6(3),6(5)-tri-O-alpha-maltosyl-beta-cyclodextrin, (G(2))(3)-betaCD] was prepared. The physicochemical and biological properties of (G(2))(3)-betaCD were determined together with those of monobranched CDs (6-O-alpha-D-glucopyranosyl-alpha-cyclodextrin (G(1)-alphaCD), 6-O-alpha-D-glucopyranosyl-beta-cyclodextrin (G(1)-betaCD), and 6-O-alpha-maltosyl-beta-cyclodextrin (G(2)-betaCD)). NMR spectra of (G(2))(3)-betaCD were measured using various 2D NMR techniques. The solubility of (G(2))(3)-betaCD in water and MeOH-water solutions was extremely high in comparison with nonbranched betaCD and was about the same as that of the other monobranched betaCDs. The formation of an inclusion complex of (G(2))(3)-betaCD with stereoisomers (estradiol, retinoic acid, quinine, citral, and glycyrrhetinic acid) depends on the cis-trans isomers of guest compounds. The cis isomers of estradiol, retinoic acid, and glycyrrhetinic acid were included more than their trans isomers, while the trans isomers of citral and quinine fit more tightly than their cis isomers. (G(2))(3)-betaCD was the most effective host compound in the cis-trans resolution of glycyrrhetinic acid. Among the branched betaCDs, (G(2))(3)-betaCD exhibited the weakest hemolytic activity in human erythrocytes and showed negligible cytotoxicity in Caco-2 cells up to 200 microM. These results indicate unique characteristics of (G(2))(3)-betaCD in some biological responses of cultured cells.     

Immunological evidence for structural homology between Drosophila melanogaster (S14), rabbit liver (S12), Saccharomyces cerevisiae (S25), Bacillus subtilis (S6), and Escherichia coli (S6) ribosomal proteins.

Specific antibodies directed against Drosophila melanogaster acidic ribosomal protein S14 were used in a comparative study of eucaryotic and procaryotic ribosomes by immunoblotting and enzyme-linked immunosorbent assays. Common antigenic determinants and, thus, structural homology were found between D. melanogaster, Saccharomyces cerevisiae (S25), rabbit liver (S12), Bacillus subtilis (S6), and Escherichia coli (S6) ribosomes.     

Three novel collagen VI chains, alpha4(VI), alpha5(VI), and alpha6(VI)

We report the identification of three new collagen VI genes at a single locus on human chromosome 3q22.1. The three new genes are COL6A4, COL6A5, and COL6A6 that encode the alpha4(VI), alpha5(VI), and alpha6(VI) chains. In humans, the COL6A4 gene has been disrupted by a chromosome break. Each of the three new collagen chains contains a 336-amino acid triple helix flanked by seven N-terminal von Willebrand factor A-like domains and two (alpha4 and alpha6 chains) or three (alpha5 chain) C-terminal von Willebrand factor A-like domains. In humans, mRNA expression of COL6A5 is restricted to a few tissues, including lung, testis, and colon. In contrast, the COL6A6 gene is expressed in a wide range of fetal and adult tissues, including lung, kidney, liver, spleen, thymus, heart, and skeletal muscle. Antibodies to the alpha6(VI) chain stained the extracellular matrix of human skeletal and cardiac muscle, lung, and the territorial matrix of articular cartilage. In cell transfection and immunoprecipitation experiments, mouse alpha4(VI)N6-C2 chain co-assembled with endogenous alpha1(VI) and alpha2(VI) chains to form trimeric collagen VI molecules that were secreted from the cell. In contrast, alpha5(VI)N5-C1 and alpha6(VI)N6-C2 chains did not assemble with alpha1(VI) and alpha2(VI) chains and accumulated intracellularly. We conclude that the alpha4(VI)N6-C2 chain contains all the elements necessary for trimerization with alpha1(VI) and alpha2(VI). In summary, the discovery of three additional collagen VI chains doubles the collagen VI family and adds a layer of complexity to collagen VI assembly and function in the extracellular matrix.     

Esterase-6 (Es-6) in laboratory mice: Hormone-influenced expression and linkage relationship to oligosyndactylism (Os), esterase-1 (Es-1), and esterase-2 (Es-2) in chromosome 8

Allelic differences at an esterase locus designated Es-6 exist between mouse strain C57 BL/6J and a laboratory stock of M.m. molossinus. Strain C57BL/6J has been assigned the allele Es-6a and M. m. molossinus the alternate allele Es-6b. Kidney expression of the electrophoretic esterase band controlled by the Es-6 locus is sex influenced, with increased activity apparently induced by testosterone. A four-point test cross established the gene order Os-Es-1-Es-6-Es-2 within a 10-cM segment on chromosome 8.     

Synthesis and coding properties of poly(c 1 A), poly(c 3 A), poly(c 7 A) and poly(h 6 A)

The homopolynucleotides poly(c¹A), poly(c³A), poly(c⁷A) and poly(h⁶A)⁺⁺ were synthesized from their corresponding nucleoside diphosphates using polynucleotide phosphorylase. With the exception of poly(h⁶A), which displayed no hypochromicity, the homopolynucleotides showed melting profiles similar to poly(A). All these polynucleotides, poly(h⁶A), poly(c⁷A), poly(c³A) and poly(c¹A) stimulated the binding of Lys-tRNA to ribosomes; the coding activity of poly(c¹A), however, was very low. Poly(h⁶A) was found to be less specific for Lys-tRNA than poly(A). The data supports the exclusive formation of Watson-Crick type base pairs and contradicts Hoogsteen base pairing in codon-anticodon recognition. Since, however, poly(h⁶A), which can form only one hydrogen bridge per base pair, stimulated the binding of Lys-tRNA comparably to poly(A), the coding activity of the homopolynucleotides tested is discussed in respect to their secondary structure as well as to the pK-values of their 6-amino groups.     

Isolation and structural analyses of positional isomers of 6(1),6(n)-di-O-(N-acetyl-beta-D-glucosaminyl)cyclomaltoheptaose (n=2, 3, and 4) and 6-O-[6-O-(N-acetyl-beta-D-glucosaminyl)-N-acetyl-beta-D-glucosaminyl]cyclomaltoheptaose.

6-O-[6-O-(N-acetyl-beta-D-glucosaminyl)-N-acetyl-beta-D-glucosaminyl]cyclomaltoheptaose (beta CD) and three positional isomers of 6(1),6(n)-di-O-(N-acetyl-beta-D-glucosaminyl)cyclomaltoheptaose (n=2, 3, and 4) in a mixture of products from beta CD and N-acetylglucosamine by the reversed reaction of beta-N-acetylhexosaminidase from jack bean were isolated and purified by HPLC. The structures of four isomers of di-N-acetylglucosaminyl-beta CDs were determined by FABMS and NMR spectroscopy. The degree of polymerization of the branched oligosaccharides produced by enzymatic degradation with bacterial saccharifying alpha-amylase (BSA) was established by LC-MS methods.     

Vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGFbeta), and interleukin-6 (IL-6) in experimental herpesvirus retinopathy: association with inflammation and viral infection

Experimental herpesvirus retinopathy presents a unique model of a transient inflammatory response in the virus-injected eye and subsequent acute retinal necrosis and chronic inflammation in the contralateral eye. For 6 days after infection, VEGF, TGFbeta1, and TGFbeta2 were associated only with inflammatory cells in the injected eye. By 6 days (after viral antigens were no longer detected), VEGF and TGFbeta2 were upregulated in retinas of injected eyes until 8-10 days. In contralateral eyes, VEGF was first demonstrated in the retina at 6-7 days (prior to the appearance of viral antigens) and TGFbeta2 at 7-8 days. Staining for these factors was also evident around areas of necrosis. The VEGF receptor, flt-1, was associated with ganglion cells and the inner nuclear layer of normal and experimental mice and it was also demonstrated around areas of necrosis. Another VEGF receptor, flk-1, was localized to Muller cell processes and the outer plexiform layer in normal and experimental mice. Coincident with VEGF upregulation in the retinas of herpesvirus-1 injected mice, there was increased flk-1 in ganglion cells and the inner and outer nuclear layers. IL-6 was associated with Muller cell endfeet in normal mice. Following unilateral intraocular inoculation, IL-6 spread along the MUller cell processes and some astrocytes demonstrated IL-6 in both eyes at 6-8 days. The present study demonstrates that intraocular inoculation of herpesvirus is sufficient to induce VEGF, flk-1, TGFbeta2, and IL-6 in the retinas of injected and contralateral eyes. Further investigation of common signaling pathways for these factors during responses to viral infection and the development of acute retinal necrosis could provide information useful for therapeutic intervention in human herpesvirus retinopathy.     

Tumour necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6) and their soluble receptors (sTNF-alpha-Rp55 and slL-6R) serum levels in systemic lupus erythematodes

We investigated a possible association between serum concentrations of tumour necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6) and their soluble receptors (sTNF-alpha-Rp55 and sIL-6R) using an enzyme-linked immunosorbent assay (ELISA) in 55 patients with systemic lupus erythematodes (SLE) and 16 healthy controls. We also examined a possible association between the serum levels of these peptides and SLE activity, as well as TNF-alpha and IL-6 concentrations and the levels of their soluble receptors. The median concentrations of TNF-alpha, sTNF-alpha-Rp55 and IL-6 were significantly higher in SLE patients than in normal individuals. In contrast, there was no difference between the serum level of sIL-6R in both groups. We found positive correlations between the serum concentrations of TNF-alpha and IL-6 as well as their soluble receptors and disease activity. There were also correlations between TNF-alpha and sTNF-alpha-Rp55 as well as IL-6 and sIL-6R serum levels in SLE patients but there were no such correlations in the normal control group. In conclusion, an increase in the serum levels of TNF-alpha, sTNF-alpha-Rp55 and IL-6 may become useful markers for SLE activity. Patients with SLE have sIL-6R serum concentration similar to that as in normal individuals. However, it correlates with disease activity and the level of IL-6.     

Herbal melanin modulates tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) production.

Recent studies have indicated that cytokines can enhance immunogenicity and promote tumor regression. However, the means for modulating cytokine production are not yet fully investigated. In this study we report the effects of a herbal melanin, extracted from Nigella sativa L., on the production of three cytokines [tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF)], by human monocytes, total peripheral blood mononuclear cells (PBMC) and THP-1 cell line. Cells were treated with variable concentrations of melanin and the expression of TNF-alpha, IL-6 and VEGF mRNA in cell lysates and secretion of proteins in the supernatants were detected by RT-PCR and ELISA. Melanin induced TNF-alpha, IL-6 and VEGF mRNA expression by the monocytes, PBMC and THP-1 cell line. On the protein level, melanin significantly induced TNF-alpha and IL-6 protein production and inhibited VEGF production by monocytes and PBMC. In the THP-1 cell line melanin induced production of all three cytokine proteins. These observations raise the prospects of using N. sativa L. melanin for treatment of diseases associated with imbalanced cytokine production and for enhancing cancer and other immunotherapies.     

Biochemical genetics of Fundulus heteroclitus (L.). III. Inheritance of isocitrate dehydrogenase (Idh-A and Idh-B), 6-phosphogluconate dehydrogenase (6-Pgdh-A), and serum esterase (Est-S) polymorphisms

Starch gel electrophoresis has shown that natural populations of Fundulus heteroclitus have variants at four enzyme-coding loci: Idh-A, Idh-B, 6-Pgdh-A, and Est-S. Analysis of the phenotypic distribution of the F₁ generation suggests that each of the variants segregates as autosomally inherited codominant alleles. Tissue specificity and intracellular localization were also determined for the IDH and 6PGDH isozymes.This work was supported by Grants DEB 76-19877 and DEB 79-12216 from the National Science Foundation and by Grant P60-80-04 from the State of Maryland. RVB and REC were supported by NIH Training Grant GM07231 to the Department of Biology.Contribution No. 1103 from the Department of Biology, The Johns Hopkins University.     

Relationship between circulating interleukin-10 (IL-10) with interleukin-6 (IL-6) type cytokines (IL-6, interleukin-11 (IL-11), oncostatin M (OSM)) and soluble interleukin-6 (IL-6) receptor (sIL-6R) in patients with multiple myeloma

We investigated the serum concentration of the interleukin-10 (IL-10), along with cytokines of interleukin-6 (IL-6) family (IL-6, IL-11 and oncostatin M - OSM), as well as soluble receptor for IL-6 (sIL-6R), in 121 patients with multiple myeloma (MM) and 28 healthy subjects. We studied the interactions between IL-10 and other cytokines, and the receptor. The correlation between IL-10 and some clinical and laboratory parameters associated with the disease activity were also analysed. The IL-10 was detectable in all patients with multiple myeloma and in all controls. The IL-10 concentration was significantly increased in myeloma patients compared with healthy persons (mean - 7.09 and 2.1 pg/ml, respectively) (p = 0.008). The level of IL-10 correlated positively with the advanced stage of disease estimated according to the Salmon and Durie classification (I versus III stage - p = 0.03). Higher values of IL-10 were found in patients with the light chain disease, hypercalcaemia, and correlated with the elevated concentrations of C-reactive protein (CRP). IL-6 was detected in 117 of the 121 patients and in all controls. The concentration of IL-6 was statistically increased in MM patients compared with control group (mean - 16.06 and 4.49 pg/ml, respectively) (p = 0.01). We found a positive correlation between IL-10 and IL-6 serum levels in MM patients. The relationship, expressed as Spearman's rank sum coefficient (rho = 0.249, p = 0.006) was significant. IL-11 was detected in 26 of the 121 MM patients and in 3 of the 28 healthy subjects at the mean concentration of 1.2 and 0.6 pg/ml respectively (p > 0.05). OSM was at detectable levels in 51 of the 121 patients and in only 4 of the 28 controls (mean - 3.84 and 0.1 pg/ml, p = 0. 002). The correlation between IL-10 and IL-11 levels in MM patients was not significant, but there was a strong statistical correlation between IL-10 and OSM concentrations (rho= 0.327, p = 0.0002). The serum concentration of sIL-6R was measurable in all patients and all controls (mean - 66.00 and 39.57 ng/ml respectively), but the difference between these groups was not significant. We found significant, positive correlation between the levels of IL-10 and sIL-6R (rho= 0.233, p = 0.01). In conclusion, we state that the serum concentrations of IL-10, IL-6, OSM and sIL-6R in MM patients may be a useful markers for the evaluation of the disease activity.     

Maternal effect and embryonic gene expression for lactate dehydrogenase B (LDH-B), glucose-6-phosphate dehydrogenase (G6PDH), and peptidase (PEP-1) during the development of Pleurodeles waltl (urodele amphibian)

The polymorphism of three enzymes [lactate dehydrogenase B (LDH-B), glucose-6-phosphate dehydrogenase, (G6PDH), and peptidase-1 (PEP-1)] in Pleurodeles waltl has allowed the expression of the corresponding loci to be followed during the development of spawnings arising from various crosses. A maternal effect lasting up to the late tail-bud stage (approx. stage 28) was shown for PEP-1. A similar situation was observed for LDH-B and G6PDH. The embryonic alleles present retarded expression: from about stage 28 for PEP-1 and G6PDH and from about stages 22 to 24 (the young tail-bud stage) for LDH-B.     

Electroanalytical investigation on the interaction between 6-mercaptopurine and zinc(II), lead(II), and cadmium(II) ions in nonaqueous solvents

Different electroanalytical techniques have been employed in the study of the interaction between 6-mercaptopurine and zinc(II), lead(II), and cadmium(II) ions in ethanol, dimethylformamide, and dimethylsulfoxide. The stoichiometry of the formed complexes was determined as well as their instability constants. The kinetic parameters of the electrode processes have been also evaluated.