Further characterization of the combining sites of Bandeiraea (Griffonia) simplicifolia lectin-I, isolectin A(4).
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Bandeiraea (Griffonia) simplicifolia lectin-I, isolectin A(4)(GS I-A(4)), which is cytotoxic to the human colon cancer cell lines, is one of two lectin families derived from its seed extract. It contains only a homo-oligomer of subunit A, and is most specific for GalNAcalpha1-->. In order to elucidate the GS I-A(4)-glycoconjugate interactions in greater detail, the combining site of this lectin was further characterized by enzyme linked lectino-sorbent assay (ELLSA) and by inhibition of lectin-glycoprotein interactions. This study has demonstrated that the Tn-containing glycoproteins tested, consisting of mammalian salivary glycoproteins (armadillo, asialo-hamster sublingual, asialo-ovine, -bovine, and -porcine submandibular), are bound strongly by GS I-A(4.)Among monovalent inhibitors so far tested, p-NO2-phenylalphaGalNAc is the most potent, suggesting that hydrophobic forces are important in the interaction of this lectin. GS I-A(4)is able to accommodate the monosaccharide GalNAc at the nonreducing end of oligosaccharides. This suggests that the combining site of the lectin is a shallow cavity. Among oligosaccharides and monosaccharides tested as inhibitors of the binding of GS I-A(4), the hierarchy of potencies are: GalNAcalpha1-->3GalNAcbeta1-->3Galalpha1-->4Galbeta 1-->4Glc (Forssman pentasaccharide) > GalNAcalpha1-->3(LFucalpha1-->2)Gal (blood group A)()> GalNAc > Galalpha1-->4Gal > Galalpha1-->3Gal (blood group B-like)> Gal. (+info)
Draculin, the anticoagulant factor in vampire bat saliva, is a tight-binding, noncompetitive inhibitor of activated factor X.
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The kinetic mechanism of action of Draculin on activated Factor X (FXa) is established. Draculin inhibits activated Factor X within seconds of incubation at near equimolar concentration (2-6 times on molar basis). Fitting the data to the equation for a tight-binding inhibitor gives a value for K(i)(K(d)) = 14.8+/-1.5 nM. The formation of the Draculin-FXa complex can be explained by a two-step mechanism, where for the first, reversible step, k(on) = 1.117 (+/- 0.169, S.E.M.) x 10(6) M(-1)s(-1) and k(off) = 15.388 (+/- 1.672) x 10(-3) s(-1), while for the second, irreversible step, which is concentration-independent, k(2) = 0.072 s(-1). K(d) obtained from k(off)/k(on) = 13.76 nM. Lineweaver-Burk plot shows a noncompetitive behavior. This noncompetitive mode of inhibition of Draculin is supported by the observation that Draculin, at concentrations giving complete inhibition, does not impair binding of p-aminobenzamidine to FXa. Moreover, under the same conditions, Draculin induces <14% decrease of the fluorescence intensity of the p-aminobenzamidine-FXa complex. We conclude that Draculin is a noncompetitive, tight-binding inhibitor of FXa, a characteristic so far unique amongst natural FXa inhibitors. (+info)
Soldier caste-specific gene expression in the mandibular glands of Hodotermopsis japonica (Isoptera: termopsidae).
(19/916)
Although "polymorphic castes" in social insects are well known as one of the most important phenomena of polyphenism, few studies of caste-specific gene expressions have been performed in social insects. To identify genes specifically expressed in the soldier caste of the Japanese damp-wood termite Hodotermopsis japonica, we employed the differential-display method using oligo(dT) and arbitrary primers, compared mRNA from the heads of mature soldiers and pseudergates (worker caste), and identified a clone (PCR product) 329 bp in length termed SOL1. Northern blot analysis showed that the SOL1 mRNA is about 1.0 kb in length and is expressed specifically in mature soldiers, but not in pseudergates, even in the presoldier induction by juvenile hormone analogue, suggesting that the product is specific for terminally differentiated soldiers. By using the method of 5'- and 3'-rapid amplification of cDNA ends, we isolated the full length of SOL1 cDNA, which contained an ORF with a putative signal peptide at the N terminus. The sequence showed no significant homology with any other known protein sequences. In situ hybridization analysis showed that SOL1 is expressed specifically in the mandibular glands. These results strongly suggest that the SOL1 gene encodes a secretory protein specifically synthesized in the mandibular glands of the soldiers. Histological observations revealed that the gland actually develops during the differentiation into the soldier caste. (+info)
Anti-arthropod saliva antibodies among residents of a community at high risk for Lyme disease in California.
(20/916)
The role of the western black-legged tick (Ixodes pacificus) versus that of other potential arthropod vectors in the epidemiology of Lyme disease was evaluated by determining the prevalence of anti-arthropod saliva antibodies (AASA) among residents (n = 104) of a community at high-risk (CHR). Salivary gland extracts prepared from I. pacificus, the Pacific Coast tick (Dermacentor occidentalis), the western cone-nose bug (Triatoma protracta), and the western tree-hole mosquito (Aedes sierrensis) were used as antigens in an ELISA. Sera from 50 residents of the San Francisco Bay region in northern California and 51 residents of Imperial County in southern California served as comparison groups. The prevalence of AASA ranged from 2% for A. sierrensis to 79% for I. pacificus in study subjects, 0% for D. occidentalis to 36% for I. pacificus among residents of the San Francisco Bay region, and 6% for I. pacificus to 24% for A. sierrensis in residents of Imperial County. The associations between AASA and demographic factors, potential risk factors, probable Lyme disease, and seropositivity for Borrelia burgdorferi were assessed for 85 members of the CHR. Seropositivity for I. pacificus and B. burgdorferi were significantly correlated, the relative risk of seropositivity to B. burgdorferi was about 5 (31% versus 6%) for subjects who were seroreactive to I. pacificus, nearly every individual who was seropositive for B. burgdorferi had elevated levels of antibodies to I. pacificus, and the mean titer for antibodies to I. pacificus was significantly higher for subjects seropositive versus those seronegative for B. burgdorferi. Together, these findings support the widely held belief that I. pacificus is the primary vector of B. burgdorferi for humans in northern California, and they demonstrate the utility of the AASA method as an epidemiologic tool for studying emerging tick-borne infections. (+info)
Candida albicans mutants deficient in respiration are resistant to the small cationic salivary antimicrobial peptide histatin 5.
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Histatins are a group of small cationic peptides in human saliva which are well known for their antibacterial and antifungal activities. In a previous study we demonstrated that histatin 5 kills both blastoconidia and germ tubes of Candida albicans in a time- and concentration-dependent manner at 37 degrees C, whereas no killing was detected at 4 degrees C. This indicated that killing activity depends on cellular energy. To test histatin 5 killing activity at lower cellular ATP levels at 37 degrees C, respiratory mutants, or so-called petite mutants, of C. albicans were prepared. These mutants are deficient in respiration due to mutations in mitochondrial DNA. Mutants were initially identified by their small colony size and were further characterized with respect to colony morphology, growth characteristics, respiratory activity, and cytochrome spectra. The killing activity of histatin 5 at the highest concentration was only 28 to 30% against respiratory mutants, whereas 98% of the wild-type cells were killed. Furthermore, histatin 5 killing activity was also tested on wild-type cells in the presence of the respiratory inhibitor sodium azide or, alternatively, the uncoupler carbonyl cyanide m-chlorophenylhydrazone. In both cases histatin 5 killing activity was significantly reduced. Additionally, supernatants and pellets of cells incubated with histatin 5 in the presence or absence of inhibitors of mitochondrial ATP synthesis were analyzed by sodium dodecyl sulfate gel electrophoresis. It was observed that wild-type cells accumulated large amounts of histatin 5, while wild-type cells treated with inhibitors or petite mutants did not accumulate significant amounts of the peptide. These data showed first that cellular accumulation of histatin 5 is necessary for killing activity and second that accumulation of histatin 5 depends on the availability of cellular energy. Therefore, mitochondrial ATP synthesis is required for effective killing activity of histatin 5. (+info)
The recombinant N-terminal region of human salivary mucin MG2 (MUC7) contains a binding domain for oral Streptococci and exhibits candidacidal activity.
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MG2 (the MUC7 gene product) is a low-molecular-mass mucin found in human submandibular/sublingual secretions. This mucin is believed to agglutinate a variety of microbes and thus is considered an important component of the non-immune host defence system in the oral cavity. We have shown that MUC7 can bind to cariogenic strains of Streptococcus mutans and that this binding requires a structural determinant in the N-terminal region. In the present study an expression construct, pNMuc7, encoding the N-terminal 144 amino acids of MUC7 was generated, and the recombinant protein rNMUC7 was expressed in Escherichia coli. Purified rNMUC7 was characterized and the binding of this protein to oral bacteria was investigated in an established assay. The results showed that the recombinant protein bound to S. mutans ATCC 25175 and ATCC 33402, and that alkylation of the two cysteine residues (Cys(45) and Cys(50)) resulted in the complete loss of bacterial binding. This suggests that binding of MUC7 to S. mutans occurs between the N-terminal region of the mucin molecule and the bacterial surface, and that this interaction is dependent on a cysteine-containing domain within this region of MUC7. In addition, the killing activity of rNMUC7 was compared with that of the candidacidal salivary protein histatin 5 in an established Candida albicans (ATCC 44505) blastoconidia killing assay. It was found that the LD(50) values of rNMUC7 and histatin 5 were comparable, and that the recombinant protein displayed significant killing activity at the physiological concentration range of MUC7 in whole saliva. This study is the first to show that the N-terminal region of MUC7 contains a structural determinant for bacterial binding and that this region exhibits candidacidal activity. (+info)
Adhesion of Candida albicans to oral streptococci is promoted by selective adsorption of salivary proteins to the streptococcal cell surface.
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Adhesion of Candida albicans to saliva-coated surfaces is an important early step in the colonization of the oral cavity. C. albicans cells also adhere to several species of oral streptococci including Streptococcus gordonii, Streptococcus oralis and Streptococcus sanguinis in what are believed to be multi-modal interactions. It is now demonstrated that incubation of streptococcal cells of these species with human parotid saliva further promotes the adhesion of C. albicans cells by up to 2-3-fold. Various species of streptococci were shown to adsorb different protein components of parotid saliva to their cell surfaces. The basic proline-rich proteins (bPRPs), to which C. albicans cells bind on nitrocellulose blot overlay, were strongly adsorbed to the surface of S. gordonii cells but not to S. oralis cells. Parotid saliva that was pre-adsorbed with S. gordonii cells and then applied to hydroxylapatite beads was <50% effective at supporting adhesion of C. albicans compared with control (non-adsorbed) saliva, demonstrating that bPRPs are major pellicle receptors. C. albicans cells did not adsorb bPRPs from fluid-phase parotid saliva. Following size-exclusion chromatography of parotid saliva samples, pooled fractions enriched in bPRPs promoted maximal adhesion of C. albicans to S. gordonii cells. The results demonstrate that C. albicans cells recognize only surface-bound forms of bPRPs and suggest that these proteins adsorbed to enamel or to streptococcal surfaces promote C. albicans adhesion and oral colonization. (+info)
The insect salivary protein, prolixin-S, inhibits factor IXa generation and Xase complex formation in the blood coagulation pathway.
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Prolixin-S is a salivary anticoagulant of the blood-sucking insect, Rhodnius prolixus, and known as an inhibitor of the intrinsic Xase. We report here its inhibitory mechanisms with additional important anticoagulation activities. We found prolixin-S specifically bound to factor IX/IXa in the presence of Ca(2+) ions. Light scattering and surface plasmon resonance studies showed that prolixin-S interfered with factor IX/IXa binding to the phospholipid membrane, indicating that prolixin-S inhibit Xase activity of factor IXa by interference with its Xase complex formation. Furthermore, reconstitution experiments showed that prolixin-S binding to factor IX strongly inhibited factor IXa generation by factor XIa. We also found that prolixin-S inhibited factor IXa generation by factor VIIa-tissue factor complex and factor IXalpha generation by factor Xa. These results suggest that prolixin-S inhibits both intrinsic and extrinsic coagulations by sequential inhibition of all coagulation pathways in which factor IX participates. It was also suggested that prolixin-S may bind to factor IX/IXa by recognizing conformational change of the Gla domain induced by Ca(2+) binding. (+info)