DNA repair profiles of disease-associated isolates of Neisseria meningitidis

Neisseria meningitidis, or the meningococcus, is the source of significant morbidity and mortality in humans worldwide. Even though mutability has been linked to the occurrence of outbreaks of epidemic disease, meningococcal DNA repair pathways are poorly delineated. For the first time, a collection of meningococcal disease-associated isolates has been demonstrated to express constitutively the DNA glycosylases MutY and Fpg in vivo. DNA sequence analysis showed considerable variability in the deduced amino acid sequences of MutS and Fpg, while MutY and RecA were highly conserved. Interestingly, multi-locus sequence typing demonstrated a putative link between the pattern of amino acid substitutions and levels of spontaneous mutagenicity in meningococcal strains. These results provide a basis for further studies aimed at resolving the genotype/phenotype relationships of meningococcal genome variability and mutator activity.     

Structure and function of threonine synthase from yeast.

Threonine synthase catalyzes the final step of threonine biosynthesis, the pyridoxal 5'-phosphate (PLP)-dependent conversion of O-phosphohomoserine into threonine and inorganic phosphate. Threonine is an essential nutrient for mammals, and its biosynthetic machinery is restricted to bacteria, plants, and fungi; therefore, threonine synthase represents an interesting pharmaceutical target. The crystal structure of threonine synthase from Saccharomyces cerevisiae has been solved at 2.7 A resolution using multiwavelength anomalous diffraction. The structure reveals a monomer as active unit, which is subdivided into three distinct domains: a small N-terminal domain, a PLP-binding domain that covalently anchors the cofactor and a so-called large domain, which contains the main of the protein body. All three domains show the typical open alpha/beta architecture. The cofactor is bound at the interface of all three domains, buried deeply within a wide canyon that penetrates the whole molecule. Based on structural alignments with related enzymes, an enzyme-substrate complex was modeled into the active site of yeast threonine synthase, which revealed essentials for substrate binding and catalysis. Furthermore, the comparison with related enzymes of the beta-family of PLP-dependent enzymes indicated structural determinants of the oligomeric state and thus rationalized for the first time how a PLP enzyme acts in monomeric form.     

Productivity-diversity relationships for plants, bryophytes, lichens, and polypore fungi in six northern forest landscapes

We investigated the relationship between site productivity and diversity of vascular plants, bryophytes, lichens, and polypore fungi in forests based on species richness data in 0.25 ha forest plots (grain size), selected from six 150–200 ha study areas (focus), and spanning over a latitudinal distance of 1350 km (extent) in Norway. We 1) searched for prevailing productivity-diversity relationships (PDRs), 2) compared PDRs among taxonomic groups and species found in different micro-habitats, and 3) investigated the effect of increasing plot (grain) size on PDRs. Using vegetation types as a surrogate for site productivity, we found a general pattern of increasing species richness with site productivity. On average total species richness doubled with a ten-fold increase in productivity. Lichens PDRs stood out as less pronounced and more variable than for other species groups investigated. PDRs of species associated with downed logs tended to level off at high-productive sites, a pattern interpreted as an effect of disturbance. Increasing the grain size >10-fold did not change the proportional difference in species richness between sites with high and low productivity.     

Critical interaction of actuator domain residues arginine 174, isoleucine 188, and lysine 205 with modulatory nucleotide in sarcoplasmic reticulum Ca2+-ATPase

ATP plays dual roles in the reaction cycle of the sarcoplasmic reticulum Ca2+-ATPase by acting as the phosphorylating substrate as well as in nonphosphorylating (modulatory) modes accelerating conformational transitions of the enzyme cycle. Here we have examined the involvement of actuator domain residues Arg174, Ile188, Lys204, and Lys205 by mutagenesis. Alanine mutations to these residues had little effect on the interaction of the Ca2E1 state with nucleotide or on the HnE 2 to Ca2E1 transition of the dephosphoenzyme. The phosphoenzyme processing steps, Ca2E1P to E2P and E2P dephosphorylation, and their stimulation by MgATP/ATP were markedly affected by mutations to Arg174, Ile188, and Lys205. Replacement of Ile188 with alanine abolished nucleotide modulation of dephosphorylation but not the modulation of the Ca2E1P to E2P transition. Mutation to Arg174 interfered with nucleotide modulation of either of the phosphoenzyme processing steps, indicating a significant overlap between the modulatory nucleotide-binding sites involved. Mutation to Lys205 enhanced the rates of the phosphoenzyme processing steps in the absence of nucleotide and disrupted the nucleotide modulation of the Ca2E1P to E2P transition. Remarkably, the mutants with alterations to Lys205 showed an anomalous inhibition by ATP of the dephosphorylation, and in the alanine mutant the affinity for the inhibition by ATP was indistinguishable from that for stimulation by ATP of the wild type. Hence, the actuator domain is an important player in the function of ATP as modulator of phosphoenzyme processing, with Arg174, Ile188, and Lys205 all being critically involved, although in different ways. The data support a variable site model for the modulatory effects with the nucleotide binding somewhat differently in each of the conformational states occurring during the transport cycle.     

A polyvalent vaccine for high-risk prostate patients: “are more antigens better?”

We have shown the immunogenicity and safety of synthetic carbohydrate vaccines when conjugated to the carrier keyhole limpet hemocyanin (KLH) and given with the adjuvant, QS-21, in patients with biochemically relapsed prostate cancer. To determine whether immune response could be further enhanced with stimulation by multiple antigens, a hexavalent vaccine was prepared using previously determined doses and administered in a Phase II setting to 30 high-risk patients. The hexavalent vaccine included GM2, Globo H, Lewis^y, glycosylated MUC-1-32mer and Tn and TF in a clustered formation, conjugated to KLH and mixed with QS-21. Eight vaccinations were administered over 13 months. All 30 patients had significant elevations in antibody titers to at least two of the six antigens; 22 patients had increased reactivity with FACS. These serologic responses were lower than that seen previously in patients treated with the respective monovalent vaccines. The reciprocal median combined IgM and IgG antibody titers with ELISA against MUC1, Tn, TF, globo H and GM2 for these 30 patients were 640, 80, 120, 40 and 0, compared to 1280, 640, 1280, 320 and 160 seen in patients receiving individual monovalent vaccines. This hexavalent vaccine of synthetic “self” antigens broke immunologic tolerance against two or more antigens in all 30 vaccinated patients, was safe, but antibody titers against several of the antigens were lower than those seen in individual monovalent trials. No impact on PSA slope was detected. We address the relevance of the multivalent approach for prostate cancer treatment. Supported by the Prostate Cancer Foundation, The PepsiCo Foundation, The Sharon Hels and Brad Reed Fund, Swim Across America, The Sara Chait Foundation. Dr. Philip Livingston is a consultant for and shareholder in Progenics Pharmaceuticals, Inc.     

The relative activities of the C2GnT1 and ST3Gal-I glycosyltransferases determine O-glycan structure and expression of a tumor-associated epitope on MUC1

In breast cancer, the O-glycans added to the MUC1 mucin are core 1- rather than core 2-based. We have analyzed whether competition by the glycosyltransferase, ST3Gal-I, which transfers sialic acid to galactose in the core 1 substrate, is key to this switch in MUC1 glycosylation that results in the expression of the cancer-associated SM3 epitope. Of the three enzymes known to convert core 1 to core 2, by the addition of GlcNAc to GalNAc in core1 C2GnT1 is the dominant enzyme expressed in normal breast tissue. Expression of C2GnT1 is low or absent in around 50% of breast cancers, whereas expression of ST3Gal-I is consistently increased. Mapping of ST3Gal-I and C2GnT1 within the Golgi pathway showed some overlap. To examine functional competition, the enzymes were overexpressed in T47D cells, which normally make core 1-based structures, have no detectable C2GnT1 activity and express the SM3 epitope. Overexpression of C2GnT1 resulted in loss of binding of SM3 to MUC1, accompanied by a decrease in the GalNAc/GlcNAc ratio, indicative of a switch to core 2 structures. Transfection of a C2GnT1 expressing line with ST3Gal-I restored SM3 binding and reduced GlcNAc incorporation into MUC1 O-glycans. Thus, even when C2GnT1 is expressed, the O-glycans added to MUC1 become core 1-dominated structures, provided expression of ST3Gal-I is increased as it is in breast cancer.     

Subtypes of non-transformed human mammary epithelial cells cultured in vitro: histo-blood group antigen H type 2 defines basal cell-derived cells

Abstract. Normal (non-transformed) human mammary epithelial cell lines derived from reduction mammoplasties were analyzed by immunocytochemistry with more than 80 monoclonal antibodies (mAbs) and other specific reagents to tissue-specific and developmentally regulated antigens at different passage levels. A subpopulation of poorly differentiated, proliferating epithelial cells, corresponding to the 'selected' cell type of late passages, is shown to be characterized by a new marker, the histo-blood group antigen H type 2, probably carried on a membrane-bound glycolipid. These cells also express a number of other onco-developmental carbohydrate antigens [Le^y, Le^x, sialosyl-Le^a, precursor of Thomsen Friedenreich antigen (T_n), but not Thomsen-Friedenreich antigen and sialosyl-T_n]. Their cytokeratin (CK) phenotype, as assessed by reactivity with monospecific mAbs and two-dimensional gel electrophoresis, is CK 5, 6, 14 and 17, with CK 19 being consistently absent, and varying minor amounts of CK 7, 8 and 18, as well as 15 and 16. The reactivity of these cells with a panel of 11 mAbs specific for CK 18 varies considerably even after cloning, indicating heterogeneity of epitope expression or accessibility. Our data strongly suggest that the H type 2⁺ cells develop from the basal cell layer of the mammary gland.     

Thymidine Kinase Diversity in Bacteria

Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria.