Protective effects of anti-antiidiotypic IgE antibodies obtained from an IgE monoclonal antibody specific for a 26-kilodalton Schistosoma mansoni antigen

A rat IgE mAb specific for larval Ag (26 kDa, 56 kDa) has been shown to protect rats against Schistosoma mansoni infection. Immunizations of Lou/M rats performed with this IgE (Ab1) induced the production of antiidiotypic antibodies (Ab2). Moreover, after this Ab2 production, anti-antiidiotypic antibodies (Ab3) were revealed. The screening of Ab3 isotypes showed the presence of IgG Ab3 and more interestingly of IgE Ab3, i.e., the same isotype as Ab1. These IgE and IgG antibodies recognized predominantly the 26-kDa Ag and were cytotoxic for schistosomula in the presence of platelets for IgE Ab3 and eosinophils for IgG Ab3. Both IgE and IgG Ab3 conferred by passive transfer protective immunity to infected rats (up to 50%). Thus the immunization with an IgE mAb led in part to the production of Ab3 of the same isotype as Ab1. In conclusion, these results suggest that the isotype selection of the antibodies of the third generation (Ab3) might be influenced by the Ab1. The respective role of the idiotope and isotype of Ab1 in isotype regulation is discussed.     

Effect of medium composition, agitation and the presence of EDTA on the antimicrobial activity of cryptolepine

The antimicrobial activity of cryptolepine is influenced by the type of medium employed, agitation and the presence of non-inhibitory concentrations of EDTA. The use of Mueller–Hinton broth (MHB), iso-sensitest broth and tryptone soya broth (TSB) produced lower minimum inhibitory concentrations (MICs) for some of the test organisms compared with nutrient broth or yeast dextrose broth (YDB). For example, a fourfold drop in MIC was recorded for Saccharomyces cerevisiae in MHB compared with the same organism tested in YDB. Agitation of the broths during incubation nearly always produced lower MICs for the bacteria, an eightfold decrease in MIC being recorded for Escherichia coli cultured in nutrient broth with agitation compared with a statically maintained culture. A non-inhibitory concentration (10⁻³ mol l⁻¹) of disodium EDTA enhanced the antimicrobial activity of cryptolepine. Against E. coli NCTC 11560, an eightfold decrease in MIC and minimum bactericidal concentration (MBC) was recorded when tested in the presence of EDTA.     

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     

Association of LHIα(B870) Polypeptide with Phospholipids During Insertion in the Photosynthetic Membrane of an LHII− Mutant of Rhodobacter capsulatus

Membranes from in vivo labeled cells of Rhodobacter capsulatus U43[pTX35] grown photosynthetically carried 60% of the [³²P]-Pi in the “heavy” fraction (HM) after sucrose gradient sedimentation. Metal-chelating chromatography of either “heavy” or “light” (LM) membrane fractions rendered similar Bchl-protein complex profiles after octyl-glucoside treatment, including most of the radioactivity in the same corresponding elution fraction (F II). Similar labeling distribution of pigment-protein complexes was obtained for membranes of dark-grown cells induced by lowering oxygen tension. Fractions derived from HM showed highly labeled LHIα, whereas the same complex from LM was essentially [³²P]-Pi-free, as revealed by SDS-PAGE followed by autoradiography. Phospholipid analysis showed a similar pattern for membranes isolated from cells photosynthetically or semiaerobically grown, being the most abundant: phosphatidylglycerol, phosphatidylethanolamine, cardiolipin, and phosphatidylcholine. Part of the phospholipids from HM comigrated with LHIα during SDS-PAGE and dissociated from the complexes only after solvent extraction and hydrophobic chromatography. However, a small amount remained always attached to LHIα, indicating an unusual strong interaction. These results suggest the existence of two operationally defined membrane regions carrying LHIα complexes differing in phosphorylation status and protein-phospholipid interaction. Received: 10 August 1996 / Accepted: 10 September 1996     

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.     

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.     

Spectrophotometric quantification of lactic bacteria in alginate and control of cell release with chitosan coating

Lactococcus lactis ssp. cremoris was entrapped within a Ca-alginate matrix, and an in situ spectrophotometric method for monitoring cell population in calcium alginate beads described. The intracapsular cell population can be estimated by measuring the optical density of beads containing cells, using cell-free beads as reference, or by measuring absorbance of a liquified bead suspension. Alginate beads, and beads coated with chitosan type I, II, and I and II mixtures, were examined for cell release. Lower viscosity chitosan (type I) coatings reduced cell release by a factor of 100 from10⁵ cfu ml⁻¹ to 10³ cfu ml⁻¹ after 6 h of fermentation. Reuse of chitosan I coated alginate beads also showed a reduction in cell release by a factor of 100. Cell loading and initial cell growth within the beads greatly affected cell release. Reducing the initial cell release would lower the overall levels of cell release throughout the fermentation. Compared to non-immobilized cultures, a 20–40% reduction in the lactic acid production rate was observed for alginate beads and chitosan I coated alginate beads, respectively. This reduction can be compensated for by increasing the intracapsular cell loading during immobilization, or before the onset of fermentation.     

A biophysical characterization of the iron coordination environment in wheat germ peroxidase

 The heme protein wheat germ peroxidase (isoenzyme C₂) and its cyanide-inhibited form have been investigated by means of electronic, CD and paramagnetic NMR spectroscopy. The data indicate a protein environment of the active site distinct from that of horseradish peroxidase (HRP), with a larger solvent accessibility. The iron is pentacoordinated at neutral and low pH, whereas a hydroxyl anion may be bound at alkaline pH. The fifth axial ligand is a His residue with a partial anionic character, as found in other peroxidases. A spin equilibrium is observed at high enzyme concentrations. Received: 17 September 1996 / Accepted: 10 January 1997