Mouse DNA methylase: methylation of native DNA.

An improved method of purification of DNA methylase from Krebs II ascites cells is reported. The enzyme sediments at 8.3 S on glycerol-gradients and a major band on SDS polyacrylamide gel electrophoresis has a molecular weight of 184 000. Aggregation occurs at low salt and this may interfere with enzymic activity. The preferred double stranded DNA substrate is that rendered partially unmethylated by an in vitro repair mechanism or by isolation from methionine starved cells. Methylation of native partially methylated DNA is favoured under conditions of low salt and high temperature; conditions which encourage 'breathing' of the DNA. Methylation of native, unmethylated DNA also involves breathing but results in formation of a salt resistant tight binding complex between the enzyme and the DNA.     

Optimum interaction of sterol side chains with phosphatidylcholine.

The specificity of the interaction between the cholesterol side chain and egg phosphatidylcholine was precisely defined by examining the effect of three new analogues of cholesterol with modified side chains on the ordering of two steroid spin labels in liposomes. The complete side chain of cholesterol was shown to be required for maximum ordering. Sterols with side chains shorter or longer than cholesterol caused significantly less ordering.     

Presence of two forms of fumarase (fumarate hydratase E.C. 4.2.1.2) in mammalian cells: Immunological characterization and genetic analysis in somatic cell hybrids. Confirmation of the assignment of a gene necessary for the enzyme expression to human chromosome 1

Two major forms of fumarate hydratase have been resolved in extracts prepared from a wide variety of mammalian cells by electrophoresis. Fractionation experiments with human and mouse cells suggest that one form (the slower migrating) is localized in the mitochondria, whereas the other form is predominant in the cytoplasm. Analysis of the segregation of the enzyme forms in human-mouse somatic cell hybrids indicates that a gene(s) necessary for the expression of both forms can be assigned to human chromosome 1 (confirmation of a previous assignment by van Someren et al., 1974). Electrophoretic analysis suggests that the two forms may be interrelated. Furthermore, they both exhibit identical reactivity toward anti-fumarate hydratase antiserum. It is suggested that a modification of one form may occur in vivo and that the modification may be important in determining the intracellular localization of the enzyme.     

Isolation and detailed characterization of human cell lines resistant to -threo-chloramphenicol

The isolation and characterization of chloramphenicol resistant derivatives of the human cell line HeLa B is described. Growth of resistant lines was unaffected in the presence of 100 μg/ml -threo-chloramphenicol, whereas growth of the parental cells was inhibited at 12.5 μg/ml. The incorporation of [³⁵S]methionine into mitochondrial protein of intact resistant cells continued normally in the presence of 100 μg/ml chloramphenicol (cytoplasmic protein synthesis was blocked by addition of 50 μg/ml emetine). Under these conditions the electrophoretic profile of labelled, presumptive mitochondrially-made proteins was similar to that of the parental cell line labelled in the absence of chloramphenicol. The cell lines selected in the presence of chloramphenicol also showed increased resistance to some other inhibitors of mitochondrial protein synthesis, e.g. carbomycin and mikamycin. [¹⁴C]Chloramphenicol was found to have normal access to the interior of resistant cells and it is therefore unlikely that resistance results from altered cell permeability. No modification of the drug by acetylation or glucuronide conjugation mechanisms was observed. The possibilities remain that resistance is mediated by altered permeability of the mitochondrial membrane, or from modification to a component of the mitochondrial protein synthetic system.     

Normalized cDNA libraries from a porcine model of orthopedic implant-associated infection

Staphylococcal infections that result from an alteration in a patient's immune response at the surgical site are a major problem in procedures that incorporate biomaterials in trauma surgery and joint replacement. Diagnosis of infection based on pathogen detection is difficult and exacerbated by increasing numbers of partially or totally resistant strains of nosocomial pathogens, particularly Staphylococcus aureus. Expression profiling of a host's cellular immune response could facilitate the identification of the pathways involved in pathogen recognition and eradication and could lead to more rational design of drugs and therapies. To this end, we constructed and characterized ten individually tagged and directionally cloned cDNA libraries from peripheral blood cells (PBC), spleen (Sp), thymus (Th), lymph node (LN), and bone marrow (BM) from immunologically naive and challenged pigs as part of an implant-associated orthopedic model of deep infection. Three of these libraries were normalized at C _{0 t} values 5, 10, 20, and 30. The libraries comprise more than 20 million primary transformants with an average insert length >1.4 kb. Cluster analysis of 7620 ESTs revealed 1029 clusters containing an average of 3.6 sequences and 3846 singletons. Gene discovery is estimated to be ∼64%. Searches of public databases resulted in 49.3% annotated porcine sequences, of which 22.2% had significant homologies to ESTs from a variety of species, and 28.5% were without a significant match in any public database. We also identified 9.1% ESTs as involved in host cell and organism defense and 11.5% related to cell signaling and communication. These sequences, together with the 28.5% appearing as novel, are of specific interest to the infectious disease process.     

Cross-immunity and antibody responses to different immobilisation serotypes of Ichthyophthirius multifiliis

Vaccination of channel catfish with either of two serotypes of the parasitic ciliate Ichthyophthirius multifiliis conferred protection against challenge infection by either serotype. Fish were vaccinated by intracoelomic injection with live theronts of isolate G5 (serotype D) or isolate G12 (a new serotype), which express different surface immobilisation antigens. Vaccination with live G12 theronts conferred complete protection against subsequent challenge by both serotypes while vaccination with G5 theronts elicited only partial protection against both serotypes. Vaccination with trophont lysates did not protect against challenge infection. Sera from vaccinated fish were tested in immobilisation assays, ELISAs, and Western blots. Serum antibodies recognised only immobilisation antigens of the serotype used for vaccination in immobilisation assays or on Western blots. No antigens common to both serotypes were identified by Western blots. In contrast, serum antibodies bound antigens in cell lysates from both serotypes by ELISA, demonstrating that antibodies recognising both serotypes are produced in response to infection, which presumably confer observed cross-serotype protection.     

Elucidation of the nature and function of the granular oyster amebocytes through histochemical studies of normal and traumatized oyster tissues

Summary A remarkable humoral component of the oyster inflammatory response was elucidated by employing the tools of the determinative histochemist. The humoral component, characterized by the release of copper and a diazotized p-nitroaniline-positive material from an acidophilic granular amebocyte, was associated with the oyster inflammatory reaction. Grossly, this humoral response was associated with the appearance of an avocado or pea green coloration in the traumatized area. A second amebocytic cell type, termed the basophilic granular amebocyte, was observed swelling in traumatized areas and may have released an additional humoral component into injured regions. Copper released in response to trauma was bound to the cells in and around the wound site and appeared to be most avidly bound by the granules of the basophilic granular amebocytes. Once incorporated into the granular matrix of these amebocytes, copper appeared to stabilize and prevent the granule from swelling.A portion of this work was excerpted from a Ph.D. thesis submitted to the Graduate School, University of Washington, Seattle.This work was supported by Public Health Service Contract No. 5 to 1 ES 00038-02. The costs of publication were defrayed in part by HSAA Award No. RR 06138 and Tumor Biology Training Grant, NIH CA 05245.     

Inhibition of mutation and combating the evolution of antibiotic resistance

The emergence of drug-resistant bacteria poses a serious threat to human health. In the case of several antibiotics, including those of the quinolone and rifamycin classes, bacteria rapidly acquire resistance through mutation of chromosomal genes during therapy. In this work, we show that preventing induction of the SOS response by interfering with the activity of the protease LexA renders pathogenic Escherichia coli unable to evolve resistance in vivo to ciprofloxacin or rifampicin, important quinolone and rifamycin antibiotics. We show in vitro that LexA cleavage is induced during RecBC-mediated repair of ciprofloxacin-mediated DNA damage and that this results in the derepression of the SOS-regulated polymerases Pol II, Pol IV and Pol V, which collaborate to induce resistance-conferring mutations. Our findings indicate that the inhibition of mutation could serve as a novel therapeutic strategy to combat the evolution of antibiotic resistance.