Human papillomavirus 6 seropositivity is associated with risk of head and neck squamous cell carcinoma, independent of tobacco and alcohol use.

BACKGROUND: The risk of head and neck squamous cell carcinoma (HNSCC) associated with common human papillomavirus types has not been well defined. METHODS: We conducted a case-control study of 1034 individuals (486 incident cases diagnosed with HNSCC and 548 population-based controls matched to cases by age, gender, and town of residence) in Greater Boston, MA. Sera were tested for antibodies to human papillomavirus (HPV)6, HPV11, HPV16, and HPV18 L1. RESULTS: HPV6 antibodies were associated with an increased risk of pharyngeal cancer [odds ratio (OR)=1.6, 1.0-2.5], controlling for smoking, drinking, and HPV16 seropositivity. In HPV16-seronegative subjects, high HPV6 titer was associated with an increased risk of pharyngeal cancer (OR=2.3, 1.1-4.8) and oral cancer (OR=1.9, 1.0-3.6), suggesting that the cancer risk associated with HPV6 is independent of HPV16. There was no association between smoking and alcohol use and HPV6 serostatus. Further, the risk of pharyngeal cancer associated with heavy smoking was different among HPV6-seronegative (OR 3.1, 2.0-4.8) and HPV6-seropositive subjects (OR=1.6, 0.7-3.5), while heavy drinking also appears to confer differing risk among HPV6-negative (OR 2.3, 1.5-3.7) and -positive subjects (OR=1.3, 0.6-2.9). CONCLUSIONS: There may be interactions between positive serology and drinking and smoking, suggesting that the pathogenesis of human papillomavirus in HNSCC involves complex interactions with tobacco and alcohol exposure.

Comparison of modular and non-modular xylanases as carrier proteins for the efficient secretion of heterologous proteins from Penicillium funiculosum.

Genes encoding three enzymes with xylanase activity from the filamentous fungus Penicillium funiculosum are described. Two of the encoded xylanases are predicted to be modular in structure with catalytic and substrate-binding domains separated by a serine and threonine-rich linker region; the other had none of these properties and was non-modular. In order to develop P. funiculosum as a host for the secreted production of heterologous proteins, each of the xylanases was assessed for use as a carrier protein in a fusion strategy. We show that one of the modular xylanases (encoded by xynA) was an effective carrier protein but the other (encoded by xynB) and the non-modular xylanase (encoded by xynC) were not effective as secretion carriers. We show that the beta-glucuronidase (GUS) protein from Escherichia coli is secreted by P. funiculosum when expressed as an XYNA fusion but that the secreted GUS protein, cleaved in vivo from XYNA, is glycosylated and enzymatically inactive.

Both binding sites of the starch-binding domain of Aspergillus niger glucoamylase are essential for inducing a conformational change in amylose.

The interaction of the two binding sites of the starch-binding domain (SBD) of Aspergillus niger glucoamylase 1 (GA-I) with substrate has been investigated by using atomic force microscopy (AFM) and UV difference spectroscopy in combination with site-specific mutants of both SBD and GA-I. The SBD possesses two binding sites with distinct affinities towards the soluble linear substrate maltoheptaose; dissociation constants (K(d)) of 17 and 0.95 microM were obtained for W563 K (binding site 2 mutant) and W590 K (binding site 1 mutant), respectively, compared to an apparent K(d) of 23 microM for the wild-type SBD. Further, the two sites are almost but not totally independent of each other for binding, since abolishing one site does not prevent the amylose chain binding to the other site. Using AFM, we show that the amylose chains undergo a conformational change to form loops upon binding to the SBD, using either the recombinant wild-type SBD or a catalytically inactive mutant of GA-I. This characteristic conformation of amylose is lost when one of the SBD binding sites is eliminated by site-directed mutagenesis, as seen with the mutants W563 K or W590 K. Therefore, although each binding site is capable of simple binding to a ligand, both sites must be functional in order to induce a gross conformational change of the amylose molecules. Taken together these data suggest that for the complex with soluble amylose, SBD binds to a single amylose chain, site 1 being responsible for the initial recognition of the chain and site 2 being involved in tighter binding, leading to the circularisation of the amylose chain observed by AFM. Binding of the SBD to the amylose chain results in a novel two-turn helical amylose complex structure. The binding of parallel amylosic chains to the SBD may provide a basis for understanding the role of the SBD in facilitating enzymatic degradation of crystalline starches by glucoamylase 1.

Function of conserved tryptophans in the Aspergillus niger glucoamylase 1 starch binding domain.

Nuclear magnetic resonance (NMR) and ultraviolet (UV) difference spectroscopy were used to assess the role of a number of tryptophan residues in the granular starch binding domain (SBD) of glucoamylase 1 from Aspergillus niger. Wild-type SBD and three variant (W563K, W590K, and W615K) proteins were produced using an A. niger expression system. Titration studies were conducted with beta-cyclodextrin (betaCD), a cyclic analogue of starch, as the ligand. The NMR studies show that the W563K and W590K variants only bind 1 equiv while the wild-type protein forms a 2:1 (ligand:protein) complex. It also clearly demonstrates the abolition of binding at site 1 and site 2 in W590K and W563K, respectively. UV difference spectroscopy was used to calculate dissociation constants with addition of betaCD: 14.4 microM (apparent) for the wild type, 28.0 microM for W563K, and 6.4 microM for W590K. The implication of this is that the two binding sites have unequal contributions to the overall binding of the SBD which may be related to functional differences between the two binding sites. The low stability of the third variant, W615K, suggests that this tryptophan is not involved in binding but has an essential structural role.

Isolation and characterisation of a linked cluster of genes from Agrobacterium tumefaciens encoding proteins involved in flagellar basal-body structure.

We report the DNA sequence of 7205 bp of the Agrobacterium tumefaciens chromosome. This contains a putative operon encoding homologues of the flagellar rod and associated proteins FlgBCG and FliE, the L and P ring proteins (FlgHI) a possible flagellum-specific export protein FliP, and two proteins of unknown function, FlgA and FliL. Several of these genes have overlapping stop and start codons. Three non-flagellate Tn5-induced mutations map to this operon: fla-11 to the first gene, encoding the rod protein FlgB; fla-15 to flgA; and fla-12 to fliL. A site-specific mutation introduced into the final gene in this cluster, fliP, also resulted in a non-flagellate phenotype. This indicates that the operon is expressed, and that at least FlgB, FlgA, FliL and FliP are required for flagellar assembly in A. tumefaciens. The bulk of this operon is conserved in the same order in Rhizobium meliloti.

The manipulation of DNA with restriction enzymes in low water systems.

The cleavage of phage lambda (lambda) DNA by the restriction enzyme HindIII in low water systems has been investigated. Two types of low water systems have been studied--those which contain a surfactant in a reverse micelle environment and a surfactant-free system in which a solid support (celite) is used. The effect of the surfactants themselves in a normal aqueous environment has also been studied. Charged surfactants were found to greatly inhibit HindIII activity in aqueous buffer, while non-ionic surfactants did not affect either the activity or the specificity of the restriction enzyme. The rate of cleavage by HindIII in a reverse micelle system consisting of sodium dioctylsulphosuccinate is very slow, however, in a Triton B system the expected fragments are observed. In a surfactant-free low water environment, cleavage occurs at the expected sites but in a different order to that observed in normal aqueous systems. These results suggest that DNA tertiary structure in low water systems is different to that in aqueous solution and that this influences cleavage by the restriction enzyme HindIII.