Molecular heterogeneity underlying the G6PD Mediterranean phenotype

Summary As part of a study aiming to define the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency, we analysed a sample from a Portugese boy with a family history of favism. Although the biochemical properties of red-cell G6PD from this subject were similar to those of the common variant G6PD Mediterranean, the corresponding mutation (563 CT) was not present. Instead, polymerase chain reaction (PCR) amplification and sequencing of the entire gene detected a CT transition at nucleotide 592 in exon VI, changing an arginine residue to a cysteine residue only 10 amino acids downstream from the Mediterranean mutation. Single-strand conformation polymorphism analysis of a PCR-amplified DNA fragment spanning exons VI and VII of the G6PD gene has detected the same mutation, confirmed by sequencing, in a G6PD-deficient patient from Southern Italy. We name this new variant G6PD Coimbra.     

Quantification of DNA uptake by Dictyostelium discoideum amoebae and the stability of the DNA during growth and development

We have developed a simple and accurate method to determine the amount of intact plasmid DNA taken up and retained by Dictyostelium discoideum amoebae during various transformation protocols. We have used this method to compare the efficiency of three different methods for introducing foreign DNA into D. discoideum amoebae. Both a calcium phosphate and a spheroplast fusion procedure gave good uptake, but no intracellular plasmid DNA was detectable after calcium chloride treatment. The exogenous DNA was rapidly lost after transformation but was 20-fold more stable during starvation rather than growth conditions, suggesting a possible approach to improving transformation efficiency. No transient expression of neomycin phosphotransferase activity of any of the heterologous animal or plant promoters used could be detected using a sensitive gel assay procedure.     

Structural studies on lamellated osmiophilic bodies isolated from pig lung. 31P NMR results and water content

1. Lamellated osmiophilic bodies are intracellular organelles in which pulmonary surfactant is stored prior to secretion. They contain about 85% phospholipid (per dry weight) and dipalmitoyl phosphatidylcholine is a major constituent, and although their ultrastructure is uncertain it is generally supposed that they resemble liposomes. However, liposomes are stable because layers of water are interposed between the lipid bilayers whereas an essential aspect of the function of lamellated bodies is that, subsequent to their secretion, they are rapidly disrupted to form a surface-active film which covers the respiratory epithelium of the lung. 2. A new method for isolating lamellated bodies from pig lung is described which has the advantage of speed and simplicity and which results in increased yields. The homogeneity of the preparation is similar to that obtained by other methods. 3. 31P NMR spectra of lamellated bodies showed that at 40 degrees C about 95% of the phospholipid was present as extended bilayers and that about 5% was present in a phase exhibiting isotropic head group mobility (tau R less than 10(-5) s). It is suggested that this phase may be due to apolar proteins which are present both in lamellated bodies and in liposomes prepared from lipids extracted from them. 4. The internal water content of lamellated bodies has been measured gravimetrically and the hydration of the phospholipid head groups has been examined by 31P NMR. The two methods gave results in good agreement and show that there are about seven molecules of water/molecule of phospholipid. It is concluded that although the phospholipid head groups in lamellated bodies are fully hydrated, there is no zone of free water. 5. Lamellated bodies are osmotically insensitive to NaCl whereas liposomes prepared from lipids extracted from them behave like perfect osmometers. It is suggested that the osmotic insensitivity and restricted water content of lamellated bodies are important to their function and dependent upon polar proteins in the outer limiting membrane.     

Magnesium-induced inner membrane aggregation in heart mitochondria: prevention and reversal by carboxyatractyloside and bongkrekic acid

Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge.     

Ligation of microglial CD40 results in p44/42 mitogen-activated protein kinase-dependent TNF-alpha production that is opposed by TGF-beta 1 and IL-10

Recently, it has been demonstrated that the CD40 receptor is constitutively expressed on cultured microglia at low levels. Ligation of CD40 by CD40 ligand on these cells results in microglial activation, as measured by TNF-alpha production and neuronal injury. However, the intracellular events mediating this effect have yet to be investigated. We report that ligation of microglial CD40 triggers activation of p44/42 mitogen-activated protein kinase (MAPK). This effect is evident 30 min posttreatment, and progressively declines thereafter (from 30 to 240 min). Phosphorylated p38 MAPK is not observed in response to ligation of microglial CD40 across the time course examined. Inhibition of the upstream activator of p44/42 MAPK, mitogen-activated protein/extracellular signal-related kinase kinase 1/2, with PD98059, decreases phosphorylation of p44/42 MAPK and significantly reduces TNF-alpha release following ligation of microglial CD40. Furthermore, cotreatment of microglial cells with CD40 ligand and TGF-beta1 or IL-10, or both, inhibits CD40-mediated activation of p44/42 MAPK and production of TNF-alpha in a statistically interactive manner. Taken together, these data show that ligation of microglial CD40 triggers TNF-alpha release through the p44/42 MAPK pathway, an effect that can be opposed by TGF-beta1 and IL-10.     

Deletion analysis of the essentiality of penicillin-binding proteins 1A, 2B and 2X of Streptococcus pneumoniae

Abstract An internal fragment from each of the penicillinebinding protein (PBP) 1A, 2B and 2X genes of Streptococcus pneumoniae , which included the region encoding the active-site serine residue, was replaced by a fragment encoding spectinomycin resistance. The resulting constructs were tested for their ability to transform S. pneumoniae strain R6 to spectinomycin resistance. Spectinomycin-resistant transformants could not be obtained using either the inactivated PBP 2X or 2B genes, suggesting that deletion of either of these genes was a lethal event, but they were readily obtained using the inactivated PBP 1A gene. Analysis using the polymerase chain reaction confirmed that the latter transformants had replaced their chromosomal copy of the PBP 1A gene with the inactivated copy of the gene. Deletion of the PBP 1A gene was therefore tolerated under laboratory conditions and appeared to have little effect on growth or susceptibility to benzylpenicillin.     

Temporal Association Between Pulmonary Inflammation and Antioxidant Induction Following Hyperoxic Exposure of the Preterm Guinea Pig

The time course and nature of the pulmonary inflammatory and antioxidant responses, both during and after hyperoxic-induced acute lung injury were studied in the preterm guinea pig. Three-day preterm (65 days gestation) guinea pigs were randomly exposed to either 21% O₂ (control) or 95% O₂ (hyperoxia) for 72 hours. All pups were then maintained in ambient conditions for up to a further 11 days, during which time lung damage was monitored. In animals exposed to hyperoxia, evidence of acute lung injury and inflammation was characterized by a marked increase in microvascular permeability and elevated numbers of neutrophils in bronchoalveolar lavage fluid. Protein concentration, elastase-like activity and elastase-inhibitory capacity in lavage fluid were at a maximum at the end of the 72 hours hyperoxic exposure. Four days later, all values had returned to control levels. In contrast, increased numbers of neutrophils, macrophages and lymphocytes were recovered in the lavage fluid during this early recovery period. Coinciding with the influx of inflammatory cells, there was a significant increase in glutathione peroxidase, manganese superoxide dismutase and catalase activities in immature lung. Lung copper/zinc superoxide dismutase activity remained unchanged during both experimental periods. The strong temporal relationship between the influx of inflammatory cells to the lung and the induction of pulmonary antioxidant enzyme defences suggests that a common mechanism underlies both responses. These findings have led us to regard inflammation in the hyperoxic-injured immature lung as a beneficial event and not, as previously suggested, as part of the injurious process.     

Biochemical maturation of the guinea pig lung and survival following premature delivery

1. Viable preterm guinea pigs were delivered by Caesarean section from 62 day gestation (term = 68 days). 2. Survival rates (24 hr) were greater than 90%, greater than 55% and greater than 35% respectively at 65, 63 and 62 days gestation. Guinea pig pups experienced increasing respiratory difficulty with progressive prematurity. 3. Lung phosphatidylcholine concentration increased steadily from 0.52 +/- 0.09 mumol/mg DNA at day 50 to 3.9 +/- 0.5 mumol/mg DNA at term. The relative contribution of the disaturated dipalmitoyl species increased over this time from 24.5 to 42.9%. 4. Pulmonary antioxidant capacity increased markedly over the final eight days of gestation, individual increases being manganese superoxide dismutase 68%, copper/zinc superoxide dismutase 48%, glutathione peroxide 37% and catalase 198%.