Search

Evidence suggests that biologically active vitamin D, 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)], may inhibit carcinogenesis. Because angiogenesis is crucial to carcinogenesis, 1,25(OH)(2)D(3) regulation of proangiogenic vascular endothelial growth factor (VEGF) secretion was investigated in cellular models for multistage carcinogenesis. Conditioned media from 1,25(OH)(2)D(3)-treated C3H10T(1/2) mouse fibroblasts and their Harvey ras-oncogene transfected counterparts (rasneo11a cells) induced human umbilical vein endothelial cell (HUVEC) proliferation (1.3 and 0.3 times, respectively, P < 0.05), suggesting that 1,25(OH)(2)D(3) altered the angiogenic phenotype of the cells. Although rasneo11a cells secreted less VEGF than C3H10T(1/2) cells (97%, P < 0.005), 1,25(OH)(2)D(3) induced C3H10T(1/2) and rasneo11a cells to secrete 2 and 3 times, respectively, more VEGF than controls (P < 0.05). Similar effects on VEGF release occurred after 1,25(OH)(2)D(3) treatment of MCF10A and MCF10Aras cells, a human breast epithelial cell model for multistage carcinogenesis. In C3H10T(1/2) cells, 1,25(OH)(2)D(3) activated the VEGF promoter in a dose-dependent (5-100 nmol/L) manner (maximum 60%) and all doses induced VEGF secretion (P < 0.05). 1,25(OH)(2)D(3) induced VEGF mRNA expression ( approximately 50%) from 2 through 24 h; VEGF release was significantly increased at 8 h and sustained for 24 h. VEGF mRNA expression and release declined as C3H10T(1/2) cells grew more confluent, whereas the magnitude of 1,25(OH)(2)D(3)-stimulated changes in VEGF was greater in confluent (3.3 times RNA; 3.5 times release) than in subconfluent (50% RNA; 100% release) cultures (P < 0.05). Thus, 1,25(OH)(2)D(3) increases VEGF secretion, and in C3H10T(1/2) cells, this is likely through activation of the VEGF promoter and induction of gene expression. These data contribute to understanding the role 1,25(OH)(2)D(3) plays in regulation of angiogenesis in normal compared with disease states.

Human salivary cystatin SN (CsnSN) is a member of the cystatin superfamily of cysteine proteinase inhibitors. In this study we used a baculovirus expression system to produce a full-length unaltered CsnSN and its variants. The variants were constructed with the changes in the three predicted proteinase-binding regions: the N-terminus (variant N(12-13), G12A-G13A), beta-hairpin loop I (variant L(56-58), Q56G-T57G-V58G) and beta-hairpin loop II (variant L(106-107), P106G-W107G). The secreted CsnSNs were purified using sequential spiral cartridge ultrafiltration and DE-52 radial flow chromatography. The purified proteins were examined for papain- and cathepsin C-inhibition. The wild-type CsnSN, and variants N(12-13) and L(106-107) bound tightly to papain (K(i) < 10 pM), whereas mutation in the loop I reduced binding affinity 5700-fold (K(i) = 57 nM). On the other hand, the wild-type CsnSN bound to cathepsin C less tightly (K(i) = 100 nM). The mutation in the N-terminus or loop I reduced binding affinity by 16 (K(i) = 1.6 microM)- and 19-fold (K(i) = 1.9 microM), respectively, while mutation in loop II resulted in an ineffective cathepsin C inhibitor (K(i) = 14 microM). Collectively, these results suggest that the N-terminal G12-G13 residues of CsnSN are not essential for papain inhibition but play a role in cathepsin C inhibition; residues Q56-T57-V58 in the loop I are essential for both papain and cathepsin C inhibitions, and residues P106-W107 in the loop II are not important for papain inhibition but essential for cathepsin C inhibition. These results demonstrated that CsnSN variants have different effects toward different cysteine proteinases., (Copyright 2000 Academic Press.)

Candida albicans is an opportunistic pathogen prevalent in AIDS patients and oral candidiasis. Azolebased drugs are currently used in the treatment of candidiasis. Histidine-rich peptides (histatins), are the natural inhibitors of candida species present in human salivary secretions. Sequence comparison of histatins revealed the common motif--KRKFHE--in active peptide fragments. Molecular modeling analysis showed structural similarity between this segment of histatins and azole-based drugs. The helical conformation adopted by histatin-5 may be stabilized by two side chain-side chain interactions (Phe... His and Arg ... Glu). Based on sequence comparison of histatin peptides and molecular modeling, a synthetic 10-residue peptide derived from histatin-5 was helical and possessed significant anti candida activity. This peptide may be used as a template to develop histatin-based drugs for treating oral candidiasis.

The lubricating properties of human submandibular-sublingual salivary fractions were examined using a servohydraulic model of mandibular movement. Fractions containing statherin exhibited a strong tendency to boundary lubrication. The lubricity of purified statherin was confirmed and compared to the amphipathic molecules gramacidin S and sodium dodecyl sulfate. Contact angle measurements of statherin paralleled the other amphipathic molecules. The helical content of statherin increased in trifluoroethanol indicating the presence of amphipathic helical regions. CD studies and hydrophobic moment calculations indicated that statherin adopts an amphipathic helical conformation at the N-terminus. An energy-minimized model of the polar N-terminal residues 1-15 suggested that this domain could be positioned in space to interact with a hydroxyapatite substrate. These data imply that under appropriate conditions statherin may display an amphipathic nature which enables it to function as a boundary lubricant on enamel.

The bioluminescent of Ca(2+)-indicator, aequorin, was loaded into the left ventricular apex of blood-perfused hearts from 13 dogs for simultaneous recording of left ventricular pressure and intracellular calcium levels. During a 2 minute period of ischemia, systolic and diastolic pressures significantly decreased. In contrast, these pressure changes were associated with an increase in both systolic and diastolic calcium reaching a maximum diastolic value of 0.59 microM and a systolic value of 1.11 microM. This apparent dissociation between pressure and [Ca2+]i supports the hypothesis that changes in myofilament Ca2+ responsiveness are of major importance in modulating contractility during ischemia in large mammalian hearts.

Previous studies have demonstrated that human salivary alpha-amylase specifically binds to the oral bacterium Streptococcus gordonii. This interaction is inhibited by substrates such as starch and maltotriose suggesting that bacterial binding may involve the enzymatic site of amylase. Experiments were performed to determine if amylase bound to the bacterial surface possessed enzymatic activity. It was found that over one-half of the bound amylase was enzymatically active. In addition, bacterial-bound amylase hydrolyzed starch to glucose which was then metabolized to lactic acid by the bacteria. In further studies, the role of amylase's histidine residues in streptococcal binding and enzymatic function was assessed after their selective modification with diethyl pyrocarbonate. DEP-modified amylase showed a marked reduction in both enzymatic and streptococcal binding activities. These effects were diminished when DEP modification occurred in the presence of maltotriose. DEP-modified amylase had a significantly altered secondary structure when compared with native enzyme or amylase modified in the presence of maltotriose. Collectively, these results suggest that human salivary alpha-amylase may possess multiple sites for bacterial binding and enzymatic activity which share structural similarities.

The effect of fluocinonide ointment and 5% crude coal tar on clearance of plaque-type psoriasis vulgaris by phototherapy was studied in 25 hospitalized patients using the bilateral comparison technique. All treated areas received ultraviolet radiation from Westinghouse fluorescent FS-40 bulbs (290-400 nm) in doses calculated to produce a minimal delayed erythema. The topically applied compounds (fluocinonide ointment, 5% Crude coal tar, white petrolatum) were applied individually or in combination. In nearly all comparisons clearance of psoriatic plaques was obtained after the same number of ultraviolet exposures, although many (8 or 14) of the areas treated with fluocinonide ointment had an accelerated early response. It thus appears that although the use of topical corticosteroids may enhance the early therapeutic response of psoriatic plaques it does not hasten the clearance of these plaques.

A blood group A+ mucin-glycoprotein was purified from aqueous extracts of rat submandibular glands by sequential chromatography on columns of Sepharose CL-6B and Sephacryl S-300 in urea-containing buffers. Final purification was facilitated by reductive methylation which appeared to release contaminating (hydrophobic) peptides. Homogeneity of the purified mucin was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at varying concentrations of acrylamide, lectin affinity chromatography, and Western blot analysis. In contrast to previously described preparations, the purified mucin contained only trace amounts of N-acetylglucosamine and aromatic amino acids. In addition, only low levels of basic amino acids were present.

The molecular cloning of sequences encoding human submandibular gland (SMG) statherin and a basic histidine-rich peptide is described. The corresponding mRNA's were highly abundant in the human and the Rhesus monkey (Macaca mulatta) SMG, but no homologous message was detectable in the murine SMG. Sequence analysis revealed strong homology between the statherin and basic histidine-rich mRNA's, suggesting their evolution from a common ancestral sequence.

A low-molecular-weight, monomeric, mucin-type glycoprotein (MG2) has been isolated from human submandibular-sublingual saliva. Initial purification involved sequential gel-filtration on Sephadex G-200 and Sepharose CL-2B, the latter in the presence of 6M urea. Fractions containing MG2 were next separated from contaminating secretory IgA by immunoaffinity chromatography or recycling through Sephadex G-200. Mucin fractions were 14C-labeled by reductive methylation, and then the final purification-step entailed recycling radiolabeled materials through Sephadex G-200. Radiolabeling aided in the assessment of purity, as judged by SDS-PAGE and ion-exchange chromatography. The carbohydrate portion accounted for 69.6% of the recovered weight and was composed of N-acetyl-glucosamine, N-acetylgalactosamine, galactose, fucose, and N-acetylneuraminic acid. Sulfate was also present. The protein comprised 30.4% of the recovered weight with threonine, serine, proline, and glycine accounting for 75.2% of the total amino acids. The oligosaccharides were alkali-labile, indicating an O-glycosyl linkage to the peptide. The mucin was weakly acidic and had an estimated mol. wt. of 200 000-250 000.

In vitro exposure of peripheral blood lymphocytes (PBLs) to doses of ultraviolet radiation (UVR) achievable during recreational sun exposure produces profound inhibition of lymphocyte blastogenesis. This study demonstrates that this inhibition is mediated by a direct dose-dependent effect on populations enriched for T-lymphocytes and is reversible after incubation of responder cells with phytohemagglutinin (PHA). In contrast, monocyte accessory function for both antigen- and mitogen-induced proliferation is relatively unaffected by in vitro exposure to UVR at doses up to 30 mJ/cm2. Exposure of PBL to UVR also results in the induction of a suppressor mechanism that inhibits the proliferation of unirradiated PBLs. In vitro exposure of human blood cells to UVR may provide a valuable tool for examining the cellular basis for the immunosuppressive effects of UVR.

A tracheobronchial mucin was isolated from the tracheobronchial secretion of a laryngectomee. It was purified by gel filtration on Sepharose CL-6B in Tris-urea buffer and rechromatography of excluded materials through the same gel matrix. It was homogeneous in 0.7% agarose-2% polyacrylamide electrophoresis under nonreducing conditions. Comparable analysis with 2-mercaptoethanol revealed at least 3 subunits. Based upon recoverable weight, the mucin was composed of 75% carbohydrate, 21% protein, and 3% sulfate. Oligosaccharides obtained by alkaline beta-elimination indicated O-glycosyl linkage to the peptide component. Marked heterogeneity of the carbohydrate side-chains was reflected in the preparation of 20 distinct oligosaccharides ranging in size from 4 to 17 residues.

A high-molecular-weight mucin-glycoprotein (MG1) was isolated from human submandibular-sublingual saliva and was comprised of 14.9% protein, 29.0% N-acetylglucosamine, 9.4% N-acetylgalactosamine, 10.5% fucose, 24.2% galactose, 0.9% mannose, 4.0% N-acetylneuraminic acid, and 7.0% sulfate. Carbohydrate units were O-glycosidically linked and ranged in size from 4 to 16 residues. The biophysical properties of MG1 were compared to those of a smaller mucin (MG2) also isolated from submandibular-sublingual saliva. Fluorescence spectroscopy demonstrated that MG1 bound both 1-anilino-8-naphthalenesulfonate (ANS) and N-phenyl-1-naphthylamine (NPNA) in stable hydrophobic binding sites (melting temperature, 47 +/- 2 degrees C), whereas MG2 did not bind these hydrophobic probes. These hydrophobic domains occurred on nonglycosylated or naked portions of MG1 since Pronase treatment eliminated ANS binding. Reduction of disulfide bridges in MG1 increased the number of available hydrophobic binding sites. High ionic strength (0 to 2 M NaCl) had no effect on ligand binding, whereas lowering pH (9 to 2) increased ANS binding without affecting NPNA complexation. Circular dichroism (CD) data suggested that MG1's carbohydrate chains dominated its spectrum. In contrast, the peptide backbone dominated the CD spectrum of MG2. Collectively, the results of this study indicate that human submandibular-sublingual saliva contains two structurally distinct mucins.