Increased resistance to anthelmintics of Haemonchus contortus eggs associated with changes in membrane fluidity of eggshells during embryonation

The embryonation of nematode eggs has been shown to increase their resistance to anthelmintics when parasites are submitted to egg hatch assays. Nevertheless, no mechanism has been suggested to explain this phenomenon. Earlier observations by other authors showed that the biochemical composition of eggshells is altered during the embryonation of eggs. The functional consequences of these changes have not been identified. We studied the changes in membrane environment (eggshells) of Haemonchus contortus eggs during the embryonation by fluidity measurements and their effects on nonspecific mechanisms of resistance to anthelmintics. We previously demonstrated that these mechanisms imply P-glycoproteins (Pgp) belonging to the multi-drug resistance (MDR) system and that the Pgp activity is very susceptible to their lipidic environment. The results obtained here show that the embryonation induced a significant and gradual increase in eggshell fluidity which was associated with an increased resistance to anthelmintics. Differences were observed between H. contortus isolates with various levels of resistance which might result from their specific biology and/or membrane composition. The membrane environment changes could act both on the solubilization of anthelmintics into the eggs and on the efflux of these lipophilic molecules by Pgp.     

Life-threatening complications of extracorporeal treatment in patients with severe eosinophilia

We report three patients with massive eosinophilia of different etiology who developed bronchoconstriction, hypotension, and shock shortly after dialysis or leukapheresis had been begun. In two cases, ethylene oxide-free materials had been used ruling out an allergic reaction related to this compound. Degranulation of eosinophils with release of eosinophil peroxidase may have caused the observed adverse reactions, as suggested by in vitro experiments with blood from the three patients. Our observations draw attention to the fact that extracorporeal therapies may initiate life-threatening complications in patients with severe eosinophilia.     

Mutation of the signal peptide region of the bicistronic gene DSPP affects translocation to the endoplasmic reticulum and results in defective dentine biomineralization

Dentine dysplasia type II is an autosomal dominant disorder in which mineralization of the dentine of the primary teeth is abnormal. On the basis of the phenotypic overlap between, and shared chromosomal location with, dentinogenesis imperfecta type II, a second disorder of dentine mineralization, it has been proposed that the two conditions are allelic. As recent studies have shown that dentinogenesis imperfecta type II results from mutation of the bicistronic dentine sialophosphoprotein gene (DSPP ), we have tested this hypothesis by sequencing DSPP in a family with a history of dentine dysplasia type II. Our results have shown that a missense change, which causes the substitution of a tyrosine for an aspartic acid in the hydrophobic signal peptide domain of the protein, underlies the phenotype in this family. Biochemical analysis has further demonstrated that this mutation causes a failure of translocation of the encoded proteins into the endoplasmic reticulum, and is therefore likely to lead to a loss of function of both dentine sialoprotein and dentine phosphoprotein.     

The disulfonic stilbene DIDS and the marine poison maitotoxin activate the same two types of endogenous cation conductance in the cell membrane of Xenopus laevis oocytes

In the present experiments we exposed the intra- or extracellular surface of excised giant membrane patches of Xenopus laevis oocytes bathed in 140 mmol/l Na-aspartate solution to the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS, 250 micromol/l). We observed that DIDS activated at least two cation conductances: (1) a non-selective cation (NSC) conductance that was mediated by channels of approximately 27 pS and resembled the stretch-activated cation conductance that has been observed in the oocyte cell membrane previously, and (2) a Na+-selective conductance, the single-channel events of which could not be resolved and which resembled the depolarization-induced Na+ conductance that has also been observed in the oocyte cell membrane previously. Both conductances were blocked by 1 mmol/l amiloride from the intra- and extracellular surfaces but inhibition of the NSC conductance by extracellular amiloride was less pronounced. Both conductances activated only slowly with a delay of 15-60 s after application of DIDS and remained active even after DIDS was washed off. This suggests that DIDS caused the exocytosis of preformed channels and this interpretation was supported by our additional observation that extracellular application of maitotoxin (MTX) mimicked the effects of DIDS. MTX is a marine toxin that has recently been reported to induce exocytosis in Xenopus laevis oocytes. The fact that DIDS and MTX each carry two sulfonyl groups suggests that they act on the same positively charged binding sites of an exocytosis-inducing protein. Our observations demonstrate that using DIDS to inhibit heterologously expressed anion transporters in the cell membrane of Xenopus laevis oocytes may compromise proper determination of the transporter currents. This effect can be prevented if the DIDS-activated endogenous cation conductances are suppressed by application of amiloride to the cytoplasmic surface of the cell membrane.     

Interaction of folate and homocysteine pathway genotypes evaluated in susceptibility to neural tube defects (NTD) in a German population

Neural tube defects (NTD) are likely to result from an interaction of several genes and environmental factors. Because periconceptional folate intake reduces the NTD risk in the fetus, and because mothers of children with NTD showed elevated plasma homocysteine levels, gene polymorphisms of the folate and homocysteine pathway, such as 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C→T, MTHFR 1298A→C and cystathionine β-synthase (CBS) 844ins68, have been implicated in the etiology of NTD. Several studies have demonstrated that these polymorphisms may indeed be associated with NTD in some populations. In order to evaluate the role of these polymorphisms and their interaction in NTD, we genotyped 417 individuals for case-control studies and 129 families for transmission disequilibrium tests. We are the first to present detailed data on MTHFR haploid genotypes in combination with CBS 844ins68. The MTHFR risk genotype 677CT/1298AC, known to be associated with decreased enzyme activity and increased homocysteine, was found significantly more often in patients than in controls (P = 0.02). A CBS insertion allele in addition to MTHFR 677CT/1298AC heterozygosity or MTHFR 677TT/1298AA homozygosity did not result in an increased risk for NTD. This is in agreement with the recently reported homocysteine-lowering effect of the CBS 844ins68 allele in carriers of MTHFR variants. Received: August 28, 2000 / Accepted: December 4, 2000     

An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow

As dendritic cells (DC) are rare populations in all organs, their generation from hematopoietic precursors in large quantities has proven critical to study their biology. From murine bone marrow about 5 x 10(6) cells at 70% purity are obtained per mouse after 8 days of culture with GM-CSF. We have improved this standard method and routinely achieve a 50-fold higher yield, i.e., 1-3 x 10(8) immature and mature DC per mouse at 90-95% purity. The major modifications were: (i) the avoidance of any active depletion of bone marrow cell subpopulations to circumvent loss of precursors, (ii) a lower plating density of bone marrow cells, (iii) a prolonged culture period of 10-12 days, (iv) the reduction of the GM-CSF dose from day 8 or 10 onwards to reduce granulocyte contaminations. The final non-adherent population at day 10-12 constitutes a mixture of immature and mature DC. Further maturation of DC could be induced by high doses of LPS or TNF-alpha for the last 24 h, where 50-70% of the non-adherent fraction represented mature DC with high levels of NLDC-145, CD86 and CD40. This method allows by simple means the generation of high numbers of murine DC with very low B cell or granulocyte contaminations. It will be valuable to study DC biology notably at the molecular level.     

Orientation of the peptidoglycan chains in the sacculus of Escherichia coli

The organization of chains of oligopeptidoglycan in the saccular wall is of critical importance in the study of the mechanism and physiology of prokaryotic wall growth. The electron microphotographs of De Pedro et al, present new findings and can be used to negate or at least raise questions about the previously accepted conclusion that the glycan chains are oriented transversely to the axis of rod-shaped Escherichia coli. This suggests caution in assuming that the glycan chains in the murein structure are parallel to each other and are perpendicular to the axis of the cell.These results should reopen the question of not only the orientation of the peptidoglycan chains, but the possibility of variability in orientation. Three classes of hypotheses about wall growth are reconsidered and problems with them are presented. The new results from De Pedro's laboratory and the experimental glycan chain length distribution argue against proposed systematic models. These include models that postulate belts or hoops stretched around the circumference of the cell and mechanisms that insert new chains of the length of presumptive “docking” strands in the stress-bearing wall. They are consistent, however, with the surface stress theory that proposes that random enzyme action together with physical forces are involved in the elongation of the rod-shaped Gram-negative wall.     

Differential blocking of coagulation-activating pathways of Limulus amebocyte lysate.

The coagulation of Limulus amebocyte lysate (LAL) can be activated through two pathways, one initiated by endotoxin and the other by beta-glucans. The two pathways join at the step of activation of the proclotting enzyme. We report here that the endotoxin-activated pathway can be differentially inhibited by two methods in a Limulus enzyme-linked immunosorbent assay (ELISA), either by the combined use of dimethyl sulfoxide and polymyxin B or by a monoclonal antibody against Limulus factor C. LAL reactivities to 10 different endotoxin preparations could be inhibited by the former method by a factor of 10(4) to 10(6) and could be blocked almost totally by the latter method, irrespective of the source of endotoxin. The sensitivity of the assay was approximately 50 pg/ml both for curdlan from Alcaligenes faecalis and for laminarin from Laminaria digitata. We also found that the beta-glucan-activated pathway could be totally blocked by laminarin (> 1 microgram/ml) without affecting the endotoxin-activated pathway, allowing endotoxin to be quantitated specifically by the Limulus ELISA with a detection limit of 0.005 endotoxin unit per ml. The use of uninhibited and differentially inhibited ELISAs demonstrated that different LAL preparations showed much greater variation in assaying beta-glucans than in assaying endotoxins. The LAL reactivity of normal human plasma was found to be due to the activation of the beta-glucan pathway, but not the endotoxin pathway, of LAL.     

A novel variety of atypical pneumocystis carinii infection after long-term prophylactic pentamidine inhalation in an AIDS patient: large lower lobe pneumocystoma

Summary Atypical pulmonary manifestations of Pneumocystis carinii infection and fair numbers of extrapulmonary and disseminated infections have lately been documented in patients with human immunodeficiency virus infection treated prophylactically with inhalative pentamidine. We report the case of a 32-year-old homosexual patient who was assessed for complaints of night sweats, weight loss, and progressive malaise. The patient denied any respiratory tract symptoms such as cough, sputum production, pleuritic chest pain, or shortness of breath. Chest X-ray revealed two large round noncavitating lesions in the lower lobe of the right lung. Pneumocystomas were diagnosed by fine-needle aspiration. A 3-week course of intravenous high-dose cotrimoxazole resulted in amelioration of symptoms but no change in the radiographic appearance of the pulmonary lesions. Four months later the patient is alive and stable and is being treated with pentamidine inhalation of 300 mg per 2 weeks and two tablets of pyrimethamine sulfadoxine per week.Abbreviations PcP Pneumocystis carinii pneumonia - Pc Pneumocystis carinii - AIDS acquired immunodeficiency syndrome - HIV human immunodeficiency virus