Molecular basis of differential B-pentamer stability of Shiga toxins 1 and 2

Escherichia coli strain O157:H7 is a major cause of food poisoning that can result in severe diarrhea and, in some cases, renal failure. The pathogenesis of E. coli O157:H7 is in large part due to the production of Shiga toxin (Stx), an AB₅ toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. There are two major isoforms, Stx1 and Stx2, which differ dramatically in potency despite having 57% sequence identity. Animal studies and epidemiological studies show Stx2 is associated with more severe disease. Although the molecular basis of this difference is unknown, data suggest it is associated with the B-subunit. Mass spectrometry studies have suggested differential B-pentamer stability between Stx1 and Stx2. We have examined the relative stability of the B-pentamers in solution. Analytical ultracentrifugation using purified B-subunits demonstrates that Stx2B, the more deadly isoform, shows decreased pentamer stability compared to Stx1B (EC₅₀ = 2.3 µM vs. EC₅₀ = 0.043 µM for Stx1B). X-ray crystal structures of Stx1B and Stx2B identified a glutamine in Stx2 (versus leucine in Stx1) within the otherwise strongly hydrophobic interface between B-subunits. Interchanging these residues switches the stability phenotype of the B-pentamers of Stx1 and Stx2, as demonstrated by analytical ultracentrifugation and circular dichroism. These studies demonstrate a profound difference in stability of the B-pentamers in Stx1 and Stx2, illustrate the mechanistic basis for this differential stability, and provide novel reagents to test the basis for differential pathogenicity of these toxins.     

Ser18 and 23 phosphorylation is required for p53-dependent apoptosis and tumor suppression

Mouse p53 is phosphorylated at Ser18 and Ser23 after DNA damage. To determine whether these two phosphorylation events have synergistic functions in activating p53 responses, we simultaneously introduced Ser18/23 to Ala mutations into the endogenous p53 locus in mice. While partial defects in apoptosis are observed in p53S18A and p53S23A thymocytes exposed to IR, p53-dependent apoptosis is essentially abolished in p53S18/23A thymocytes, indicating that these two events have critical and synergistic roles in activating p53-dependent apoptosis. In addition, p53S18/23A, but not p53S18A, could completely rescue embryonic lethality of Xrcc4(-/-) mice that is caused by massive p53-dependent neuronal apoptosis. However, certain p53-dependent functions, including G1/S checkpoint and cellular senescence, are partially retained in p53(S18/23A) cells. While p53(S18A) mice are not cancer prone, p53S18/23A mice developed a spectrum of malignancies distinct from p53S23A and p53(-/-) mice. Interestingly, Xrcc4(-/-)p53S18/23A mice fail to develop tumors like the pro-B cell lymphomas uniformly developed in Xrcc4(-/-) p53(-/-) animals, but exhibit developmental defects typical of accelerated ageing. Therefore, Ser18 and Ser23 phosphorylation is important for p53-dependent suppression of tumorigenesis in certain physiological context.     

Immunocytochemical localization of heparan sulfate proteoglycan in human decidual cell secretory bodies and placental fibrinoid

A distinct ultrastructural feature of human decidual cells is the presence of membrane-bound secretory bodies, 0.3-0.5 micron in diameter, located within club-shaped processes at the cell periphery. These secretory bodies contain 30-60 nm electron-dense granules. Using specific antibody and the protein A-gold technique, we examined the localization of heparan sulfate proteoglycan in human decidual cells. Morphometric analysis of gold particles in cellular compartments was performed with a Zeiss Videoplan computer system. Immuno-gold staining was present in the decidual cell cytoplasm and the extracellular space, especially in the zone of the external lamina. Gold particles, indicating the locale of heparan sulfate proteoglycan, were concentrated over the electron-dense granular material within decidual secretory bodies contained in club-shaped processes at the cell periphery. Immunolabeling of placental fibrinoid was also observed. This report provides the first identification of a specific molecular constituent of decidual secretory bodies and indicates a role for these structures in secretion of the peri-decidual cell extracellular matrix.     

Carrier detection in haemophilia B using two further intragenic restriction fragment length polymorphisms.

A normal human population has been screened for the existence of further restriction fragment length polymorphisms (RFLPs) in the clotting factor IX gene in addition to the TaqI polymorphism already characterised (1,2). Two polymorphic loci were found, both within 6 Kb of the TaqI polymorphism within the body of the factor IX gene. One of the polymorphisms has been shown to be due to either the presence or absence of a particular recognition site for the restriction enzyme XmnI. The other, visualised as a difference in fragment pattern produced by digestion with either HinfI or DdeI, has two allelic forms differing by a 50 bp element of inserted DNA. Sequence analysis has shown the inserted element to be in a region of Z type DNA sequence, the insertion representing a duplication of flanking sequence on either side. The two polymorphisms are inherited in simple Mendelian fashion and have both been used to diagnose haemophilia B carrier status. It is estimated that the combined use of these polymorphisms in the factor IX gene, despite linkage disequilibrium between the 3 polymorphic loci, should enable carrier status to be determined in approximately 66% of all haemophilia B families.     

Activation of mutated simian virus 40 enhancers by amplification of wild-type enhancer elements.

We show that duplication of any one of three separate simian virus 40 enhancer elements, A, B, or C, can compensate for loss of function in the remaining two. Simian virus 40 revertants containing point mutations within the A and C (dpm16) or B and C (dpm26) enhancer elements contain tandem duplications that include the remaining wild-type element. These simple tandem duplications can create enhancers 25-fold more active than that of the parental mutant. These revertants can arise by illegitimate recombination between heterologous viral genomes. This was demonstrated by the recombinants resulting from a mixed infection with the viruses dpm16 and dpm2, which contain mutations in the A and C elements and the B element, respectively.     

Human seminal vesicle-specific antigen during semen liquefaction

Binding of monoclonal antibody MHS-5 to western blots of human seminal plasma was employed to follow the fate of a seminal vesicle-specific antigen (SVSA) during semen liquefaction. Ejaculates from four vasectomized donors were collected in a manner to inhibit liquefaction or to allow liquefaction to proceed at room temperature. Aliquots of the liquefying seminal fluid were removed at specific time points and further liquefaction inhibited with sodium dodecyl sulfate (SDS). Western blot analysis using monoclonal antibody MHS-5 demonstrated that the SVSA epitope in all donors was located on multiple bands ranging in mass from 15-92 kDa in unliquefied ejaculates; immunoreactive peptides below 15 kDa were not detected. As early as 5 min post ejaculation, immunoreactive bands below 15 kDa were identified in liquefying samples. During the same time period (5 min), immunoreactive bands of 69-71 and 58 kDa could not be immunologically detected in liquefying samples. A decrease in immunoreactive staining of components higher molecular mass was accompanied by a concomitant increase in immunoreactive staining of intermediate and small molecular mass molecules during the first 2 h of liquefaction. After 8-24 h of liquefaction, two immunoreactive bands of 10.9 and 12.5 kDa predominated. Between 24 and 48 h, each donor's ejaculate demonstrated a common single immunoreactive band of 10.9 kDa. These results indicate that there is a rapid transformation in mass of the SVSA with major 69-71 and 58 kDa bands being converted to forms of lower mass within 5 min of ejaculation.     

Purification and properties of an extracellular β-glucosidase from Lenzites trabea

Summary When culturing the cellulolytic-active Basidiomycete and brown-rot fungus Lenzites trabea A-419 in submerged culture with glucose and cellulose as a carbon source, the fungus only excreted -glucosidase (EC 3.2.1.21) and an endo-1,4--glucanase (EC 3.2.1.4).No evidence for C₁ activity (EC 3.2.1.91) was found in the culture filtrate or in the ultra concentrate. -Glucosidase could be separated from endoglucanase by chromatography on Sepharose 6-B. Further fractionation of the -glucosidase on DEAE-Sephadex A-25 resulted in a 525-fold purification. The molecular weight of the isolated -glucosidase was determined by co-chromatography on Sephadex G-200 to be 320,000 daltons. The enzyme developed maximum activities at pH 4.5 and 75°C. The enzyme does not act on crystalline cellulose or CMC, but it hydrolyzes cellotriose,-tetraose, and-pentaose to cellobiose and glucose. -glucosidase activity was strongly inhibited by the reaction product, glucose. A K_i value of 2.7×10^–3 (M) for noncompetitive inhibition was found.     

Purification and properties of an extracellular endo-1,4-β-glucanase fromLenzites trabea

An extracellular endo-1,4--glucanase (EC 3.2.1.4) has been isolated and purified from the culture solution of the basidiomyceteLenzites trabea grown on glucose and cellulose. Besides-glucosidase activity (EC 3.2.1.21) no evidence for C₁-activity (EC 3.2.1.91) in the culture solution was found.The endoglucanase has been purified in a four-step procedure including chromatography on Sepharose 6-B and DEAE-Sephadex A-50, adsorption on hydroxylapatite and gel filtration on Bio-Gel P-100. The enzyme showed maximum activity at pH 4.4 and 70°C. A molecular weight of 29000 Daltons was estimated by calibration on Bio-Gel P-100. The enzyme hydrolyses carboxymethyl cellulose (CMC) as well as xylan.List of Abbreviations CMC carboxymethyl cellulose - D.S. degree of substitution - D.P. degree of polymerisation - MW molecular weight     

Mechanisms for flexibility in DNA sequence recognition and VP16-induced complex formation by the Oct-1 POU domain.

DNA binding by the Oct-1 protein is directed by its POU domain, a bipartite DNA-binding domain made up of a POU-specific (POUS) domain and a POU-homeo (POUH) domain, two helix-turn-helix-containing DNA-binding modules that cooperate in DNA recognition. Although the best-characterized DNA target for Oct-1 binding is the octamer sequence ATGCAAAT, Oct-1 also binds a number of different DNA sequence elements. For example, Oct-1 recognizes a form of the herpes simplex virus VP16-responsive TAATGARAT element, called the (OCTA-)TAATGARAT site, that lacks octamer site similarity. Our studies suggest two mechanisms by which Oct-1 achieves flexible DNA sequence recognition. First, an important arginine found in the Oct-1 POUS domain tolerates substitutions of its base contacts within the octamer site. Second, on the (OCTA-)TAATGARAT site, the POUS domain is located on the side of the POUH domain opposite from where it is located on an octamer site. This flexibility of the Oct-1 POU domain in DNA binding also has an impact on its participation in a multiprotein-DNA complex with VP16. We show that Oct-1 POUS domain residues that contact DNA have different effects on VP16-induced complex formation depending on whether the VP16-responsive element involved has overlapping octamer similarity or not.