Mycobacterium tuberculosis complex DNA in calcified pleura from remains 1400 years old

Mycobacterium tuberculosis complex DNA was isolated and identified in calcified pleura from remains 1400 years old, with the polymerase chain reaction. This is the first demonstration of tuberculosis in non-mummified archaeological tissue other than bone; the presence of mycobacterial mycolic acids in the sample supports this conclusion. The study of ancient DNA from microbial pathogens is of interest as it enables verification of traditional diagnoses, may answer long-standing questions in the history of disease, and provides ancient DNA sequences that can be compared with those of modern isolates.     

An improved bacteriophage lambda vector: construction of model recombinants coding for kanamycin resistance.

An attenuated bacteriophage lambda has been prepared for proposed use as an EK2 vector. This phage, designated lambdagt vir Jam27 Zam718-lambdaB' can accomodate up to 11.10(6) daltons of foreign DNA inserted through Eco RI ends. The virulence mutations and nin 5 reduce the frequency of lysogen and/or plasmid formation. The mutations Jam27 and Zam718 require a suppressor in the bacterial host. The phage recombination functions contained in the EcoRIlambdaC fragment have been deleted, and only the EcoRIlambdaB fragment remains (in reverse orientation) in the center portion of the vector. In addition, this phage adsorbs to sensitive bacteria at a significantly reduced rate, conferring another block to the escape of free phage. Model recombinants have been constructed by in vitro recombination with an EcoRI fragment coding for kanamycin resistance (originally derived from R-factor R6-5). This fragment of DNA is 4.6.10(6) daltons in size, contains an inverted repeat, and also appears to contain a promoter for the kanamycin resistance gene. Using this model recombinant, the rate of transfer of kanamycin resistance to permissive and nonpermissive strains of E. coli has been measured.     

Biosynthesis of the v-sis gene product: signal sequence cleavage, glycosylation, and proteolytic processing.

The v-sis oncogene and its cellular homolog c-sis encode chain B of platelet-derived growth factor. Cells transformed by v-sis produce a platelet-derived growth factor-related molecule which is able to stimulate the platelet-derived growth factor receptor in an autocrine fashion. Site-directed mutagenesis was used to construct several mutations which substitute charged residues for hydrophobic residues in the proposed signal sequence of the v-sis gene product. Two of these mutations resulted in the synthesis of altered v-sis gene products with an unexpected nuclear location and a loss of biological activity. We also report here the intracellular localization of the v-sis gene product to the endoplasmic reticulum-Golgi compartment, where signal sequence cleavage and N-linked glycosylation occur. The v-sis gene product contains no transmembrane regions, as it is completely protected within isolated microsomes from trypsin proteolysis. Site-directed mutagenesis was also used to alter a proposed proteolytic processing site in the v-sis gene product. This mutant v-sis gene, which encodes Asn-Ser in place of Lys-Arg at residues 110 to 111, was found to retain full biological activity.     

Effect of inoculation sequence and nutrients upon Streptococcus mutans BHT and Streptococcus mitior LPA-1 growing on human teeth in an artificial mouth

Human teeth in an artificial mouth were inoculated with Streptococcus mutans BHT, Streptococcus mitior LPA-1, or sequentially with both organisms. Incubation was continued for 90 h. Mixed populations were largest when a nutrient supplement containing 5–0% (w/v) sucrose was supplied. Fewer organisms were recovered from experiments with synthetic saliva only, or when a supplement containing 0–05% (w/v) glucose was available. The inoculation sequence determined the total viable count and a larger population resulted when Strep, mutans was the initial colonizer ( P < 0–01). Strep, mutans was always able to become established even when super-infected on to a 24 h plaque of Strep, mitior. The final proportion of Strep mutans was lower when it was the superinfecting organism and the sucrose ( P < 0–01) or glucose ( P < 0–05) nutrient supplement was provided. This work confirms the importance of inoculation sequence and presence of sugars in plaque accumulation and demonstrates the fundamental role of microbial interactions in this process.     

Glycoconjugates of the human trabecular meshwork: a lectin histochemical study

Summary Twelve specimens of resin-embedded human trabecular meshwork were probed with a panel of 21 biotinylated lectins, using an avidin-biotin peroxidase revealing system, in order to determine the normal pattern of saccharide expression in this tissue. High-mannose, intermediate and hybrid N-linked glycans, and complex N-linked bisected and non-bisected bi/tri-antennate glycans, as shown by the binding ofCanavalia ensiformis (ConA),Pisum sativum (PSA),Lens culinaris (LCA) agglutinins andPhaseolus vulgaris erythroagglutinin (ePHA), were strongly expressed by the canal of Schlemm endothelium and juxtacanalicular tissue, but less so by the corneoscleral meshwork. Highly branched complex glycans were not found, as there was no binding byPhaseolus vulgaris leukoagglutinin (1PHA). Sialyl residues, especially thoseα2,6-linked as demonstrated by strongSambucus nigra (SNA) lectin staining, were also abundant in this area.N-acetyllactosamine sequences and some O-linked glycans were present in the trabecular meshwork, as shown bySolanum tuberosum (STA),Datura stramonium (DSA), andJacalin (Jac) lectin binding, while fucose residues were not detected byTetragonolobus purpureas (LTA) orUlex europaeus-1 (UEA-1) agglutinins. These results indicate similarities with renal glomerular and vascular endothelium, although the lack of binding with UEA-1 agglutinin suggests differences which may relate to the specialized function of the trabecular meshwork. This study provides a baseline for comparative analysis of the glycans of human trabecular meshwork in pathological conditions such as primary open-angle glaucoma.