Phorbol ester-stimulated superoxide production by murine bone marrow-derived macrophages requires preexposure to cytokines

Murine resident peritoneal macrophages (RPM) generate superoxide (O2-) in response to stimulation with PMA or zymosan. Murine bone marrow-derived macrophages (BMM) generate O2- in response to zymosan but not PMA. However, the ability to generate O2- in response to PMA could be induced in BMM by pre-exposing the cells to certain cytokines, including granulocyte-macrophage CSF (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), IFN-gamma, and, to a lesser extent, IL-1 alpha. Bacterial LPS also induced the ability to respond to PMA. These same agents were also shown to prime RPM for enhanced PMA-induced respiratory burst. In contrast to GM-CSF, CSF-1 did not enhance the ability of BMM or RPM to generate O2- in response to PMA. Pretreatment with GM-CSF or TNF-alpha did not significantly affect the zymosan-induced release of O2- by BMM. These results suggest that unprimed BMM have a deficiency in the PMA-dependent signaling pathway that is corrected by exposure to selected cytokines. The results also raise the possibility that the basal ability of tissue macrophages to generate a respiratory burst in response to PMA may be a reflection of in vivo exposure to cytokines.     

Some new variants of serum protease inhibitors in Meishan pigs

Serum samples of Meishan (13 animals) and Meishan x Wild Boar crosses (361 animals) were analysed by means of two-dimensional electrophoresis. Some new variants in protease inhibitor systems PO1A, PO1B and PI2 are reported.     

Detection and characterization of mitochondrial DNA rearrangements in Pearson and Kearns-Sayre syndromes by long PCR

We used a strategy based on long PCR (polymerase chain reaction) for detection and characterization of mitochondrial DNA (mtDNA) rearrangements in two patients with clinical signs suggesting Pearson syndrome and Kearns-Sayre syndrome (KSS), respectively, and one patient with myopathic symptoms of unidentified origin. Mitochondrial DNA rearrangements were detected by amplification of the complete mitochondrial genome (16.6 kb) using long PCR with primers located in essential regions of the mitochondrial genome and quantified by three-primer PCR. Long PCR with deletion-specific primers was used for identification and quantitative estimation of the different forms of rearranged molecules, such as deletions and duplications. We detected significant amounts of a common 7.4-kb deletion flanked by a 12-bp direct repeat in all tissues tested from the patient with Pearson syndrome. In skeletal muscle from the patient with clinical signs of KSS we found significant amounts of a novel 3.7-kb rearrangement flanked by a 4-bp inverted repeat that was present in the form of deletions as well as duplications. In the patient suffering from myopathic symptoms of unidentified origin we did not detect rearranged mtDNA in blood but found low levels of two rearranged mtDNA populations in skeletal muscle, a previously described 7-kb deletion flanked by a 7-bp direct repeat and a novel 6.6-kb deletion with no repeat. These two populations, however, were unlikely to be the cause of the myopathic symptoms as they were present at low levels (10–40 ppm). Using a strategy based on screening with long PCR we were able to detect and characterize high as well as low levels of mtDNA rearrangements in three patients. Received: 10 March 1997 / Accepted: 20 May 1997     

Expression of mosquitocidal crystal protein genes in non-insecticidal Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis NTB-1 isolated from soil samples in Korea produces ovoidal parasporal inclusions with proteins of approximately 24–40 kDa in size. Although serological study indicated that the isolate has a flagella (H) antigen identical with subsp. israelensis , it seemed to be non-insecticidal against Lepidoptera and Coleoptera as well as Diptera. To investigate the activity of non-insecticidal B. thuringiensis transformed with insecticidal crystal protein genes, cryIVD and cytA genes of B. thuringiensis subsp. morrisoni PG-14, highly toxic to mosquito larvae, were introduced into the isolate NTB-1. The expression of mosquitocidal crystal protein genes in NTB-1 was characterized by SDS–PAGE analysis and electron microscopy. The results showed that crystalline inclusions of host, CryIVD and CytA were stably expressed in the transformant. However, the mosquitocidal activity of transformant was similar to that of B. thuringiensis subsp. kurstaki Cry⁻ B harbouring cryIVD and cytA genes, demonstrating that a synergistic effect by an interaction of both introduced insecticidal and resident non-insecticidal crystal proteins was not observed.     

An immuno-dot blot assay for detection of thermostable protease from Pseudomonassp. AFT-36 of dairy origin

A dot-ELISA technique for the detection of Pseudomonas protease was developedusing IgG of anti- Pseudomonas AFT-36 protease as capture antibody. The detection limitof protease in buffer or milk was 1·01 ng ml⁻¹. The procedure was performedat room temperature, took about 2·5 h and was economical. Protease AFT-36 isimmunologically related to five out of seven Pseudomonas spp. The results suggest thatthe assay could be used to detect proteases in dairy products.     

The incidence of Weeksella- and Bergeyella-like bacteria in the food environment

Eighty-five catalase- and oxidase-positive Gram-negative rods and cocci susceptible to penicillin G were isolated from a variety of food sources. The phenotypic relationships of these isolates with reference cultures of Bergeyella -like, Chryseobacterium, Empedobacter, Myroides , Moraxella , Sphingobacterium and Weeksella -like strains were examined by numerical taxonomy. Seventy-three isolates were recovered in five groups; 80% of the isolates clustered in groups 1, 2 and 3 and produced indole, bearing a strong resemblance to Weeksella and Bergeyella . They could not, however, be regarded as belonging to the known species of W. virosa and B. zoohelcum . It is suggested that three species may be necessary to accommodate the environmental Weeksella - or Bergeyella -like bacteria. The isolates in groups 4 and 5 had white colonies and were unable to produce indole, in this way resembling the Moraxella genus.     

Mutational analysis of slyD, an Escherichia coli gene encoding a protein of the FKBP immunophilin family

slyD encodes a 196 amino acid polypeptide that is a member of the FKBP family of cis–trans peptidyl–prolyl isomerases (PPIases). slyD mutations affect plaque formation by the phage φX174 by blocking the action of the phage lysis protein E. Here we describe the selection of a set of spontaneous slyD mutations conferring resistance to the expression of gene E from a plasmid. These mutations occur disproportionately in residues of SlyD that, based on the structure of the prototype mammalian FKBP12, make ligand contacts with immunosuppressing drug molecules or are conserved in other FKBP proteins. A wide variation in the plating efficiency of φX174 on these E  ^R strains is observed, relative to the parental, indicating that these alleles differ widely in residual SlyD activity. Moreover, it is found that slyD mutations cause significant growth rate defects in Escherichia coli B and C backgrounds. Finally, overexpression of slyD causes filamentation of the host. Thus, among the FKBP genes found in organisms across the evolutionary spectrum, slyD is unique in having three distinct drug-independent phenotypes.