Binding and aggregation of the 25-kilodalton toxin of Bacillus thuringiensis subsp. israelensis to cell membranes and alteration by monoclonal antibodies and amino acid modifiers

The 25-kilodalton toxin of Bacillus thuringiensis subsp. israelensis binds irreversibly to Aedes albopictus cells, Choristoneura fumiferana cells, and erythrocytes. The binding to cells increased with both toxin concentration and time and when the cells were first preincubated with unlabeled toxin. Binding data indicated a two- to threefold increase in the rate of binding after the amount of the membrane-bound toxin reached approximately 3.5 fmol/3 x 10(5) A. albopictus cells or 3.3. fmol/2 x 10(5) C. fumiferana cells. When this level of bound toxin was reached, the toxins also began forming aggregates at the cell membrane. The toxin aggregates were extracted with 10% Triton X-100 and separated from the monomers with a 5 to 20% sucrose density gradient. The toxin aggregates isolated from A. albopictus and C. fumiferana cell membranes were ca. 400 kilodaltons, while those isolated from human erythrocytes were significantly smaller. The proportion of the toxin found in aggregate form increased rapidly with the amount of toxin bound; however, the molecular size of the aggregates remained constant. Eleven monoclonal antibodies raised against the native form of the toxin blocked 80 to 97% of the toxin binding to cells. The epitope of one of these monoclonal antibodies was mapped to a domain which included the cysteine, suggesting the importance of the domain around this amino acid to binding. Toxin binding and cell lysis were also inhibited by treating the toxin with HgCl2, further indicating the importance of the C-terminal hydrophobic cysteine-containing domain in cytolytic activity of the 25-kilodalton protein.     

The analysis of hypervariable DNA profiles: problems associated with the objective determination of the probability of a match

Summary A computerised system has been used to store DNA profiles from 3 hypervariable loci. This initial survey illustrates that band matching is only possible after analysis of the errors associated with electrophoretic systems. A number of databases have been constructed with the three probes investigated and two methods of frequency determination, binning and sliding window fitting, have been compared.     

Histopathology and cell culture characteristics of liver cells from grc − and grc + rats given diethylnitrosamine

The histopathological response and cell culture characteristics of liver cells from the R16 (grc ^–) strain of rats, which carries an MHC-linked deletion, were examined one week after a single intraperitoneal injection of 200 mg/ kg body weight diethylnitrosamine (DEN) and were compared with the response of liver cells from wild type (grc+) rats. The DEN exposure induced hydropicl vacuolar changes in the parenchymal cells and a limited proliferation of oval cells in the periportal areas of the livers of both grc+ and grc rats. Primary culture of collagenase-digested livers consisted of parenchymal, bile ductular and oval-related cells as determined by cell-specific immunohistochemistry. Subpassaged cells from grc+ rats exhibited oval cell ultrastructural morphology, inducible histochemical staining for gammaglutamyl transpeptidase (GGT), and DEN-associated onset of anchorage-independent growth. Primary cultures of liver cells from R16 rats consistently failed to form cell strains upon subpassage.Abbreviations DEN diethylnitrosamine - grc growth and reproduction complex - GGT gamma-glutamyl transpeptidase - MHC major histocompatibility complex     

Frequent duplication and deletion events in the 5S RNA genes and the associated spacer regions of theTriticeae

The 5 S DNA units from 15 grasses in theTriticeae were analysed at the DNA sequence level. Four units carried duplications near the 3-end of the 5 S RNA gene with 3 of the duplications centred on the same base pairs as a duplication previously reported byGerlach & Dyer. The fourth duplication was located 3 downstream from the gene, in the spacer region. Apparent deletions were very frequent when units of the different grasses were compared and it was clear that these deletions did not extend into a 75 bp spacer region upstream from the 5 S RNA gene. This 75 bp region also tended to be more conserved between the grasses as compared to the high level of sequence change in the rest of the spacer region. — Phenetic relationships were established between the grasses using the sequence data. The relationships were generally consistent with the data from other parameters and, in addition, showed that two Australian grasses were closely related to the other Northern hemisphere genera examined. The data concerning the Australian grasses is discussed in relation to the isolated nature of Australia.     

Ligand-induced endocytosis of the EGF receptor is blocked by mutational inactivation and by microinjection of anti-phosphotyrosine antibodies

Early events in ligand-induced endocytosis of the EGF receptor have been examined. A mutant EGF receptor devoid of intrinsic protein-tyrosine kinase activity bound EGF and dimerized normally yet failed to undergo ligand-induced internalization. Immunofluorescence microscopy revealed that receptors lacking kinase activity failed to undergo the ligand-induced internalization characteristic of receptors with kinase activity. Monoclonal anti-phosphotyrosine antibodies effectively inhibited phosphorylation of exogenous substrates in vitro and, when microinjected into cells containing active EGF receptors, prevented internalization of the receptor when cells were subsequently challenged with EGF. These results point to a crucial role for the kinase activity of the EGF receptor in the process of ligand-induced endocytosis of receptors, and imply that a phosphorylated substrate(s) is required.     

Effect of ciprofloxacin on subcutaneous abscesses induced with Staphylococcus epidermidis and a foreign body implant in the mouse

Subcutaneous abscesses were induced in mice with Staphylococcus epidermidis strain G19-85 and a foreign body implant. The MIC of ciprofloxacin for this strain was 0.25 microgram/ml. The ciprofloxacin dosage, 120 mg/kg/day, was divided into three injections, administered to the mice subcutaneously at 8 h intervals. Serum concentration kinetics in normal mice (n = 50) were determined. The peak serum level of ciprofloxacin was 3.18 micrograms/ml at the 15 min sampling time; the trough level was 0.53 micrograms/ml at 8 h. Abscesses were found in 96% (n = 49) of the untreated, infected control mice. Three modes of treatment with ciprofloxacin were tested: (1) four prophylactic injections of ciprofloxacin prior to infection reduced abscess formation to 64% (p less than or equal to 0.0002, n = 50). (2) Eleven therapeutic injections, initiated 4 days after infection, reduced abscess formation to 86% (p less than or equal to 0.17, n = 49). (3) One prophylactic injection prior to surgery and five therapeutic injections after infection reduced abscess formation to 43% (p less than or equal to 0.0001, n = 49). Culture results correlated with the abscess formation rates.     

Poly(ADP-ribose) degradation by glycohydrolase starts with an endonucleolytic incision

We have recently shown that poly(ADP-ribose) polymerase forms poly(ADP-ribose) by adding ADP-ribose residues to the polymerase-proximal end of an enzyme-bound nascent chain. In this light we have reexamined the mode of hydrolysis of enzyme-bound poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase. When the substrate has been labeled by a pulse-chase protocol, soluble glycohydrolase releases a significant amount of labeled oligomer which can only come from the enzyme-distal (2') end of the polymer. This constitutes additional evidence for the proximal growth of chains. Oligomer is infrequently released from the proximal (1") end of enzyme-bound chains. Rather, the bulk of the poly(ADP-ribose) is digested directly to ADP-ribose monomers. We conclude that poly(ADP-ribose) glycohydrolase starts digestion with an endonucleolytic incision and then removes ADP-ribose residues processively in the 2'----1" direction. Therefore, in contrast to earlier models of polymer growth and hydrolysis, a single poly(ADP-ribose) chain may be extended at one end and simultaneously degraded at the other end. The balance between synthesis and degradation may control the quantity and distribution of polymer around the DNA break which occasions its synthesis.