Severe non-pneumonic necrotising infections in children caused by Panton-Valentine leukocidin producing Staphylococcus aureus strains.

Two cases of infection with Panton-Valentine Leukocidin (PVL) producing strains of Staphylococcus aureus are reported. A 15-year-old insulin dependent diabetic developed toxic shock syndrome and an abscess in the deep tissue around his left hip. A 34-day-old infant presented with a right orbital cellulitis with an intra-orbital collection and septicaemia. In both cases PVL-producing strains of Staphylococcus aureus were isolated. Both surgery and prolonged antibiotic combination regimens were required to eradicate the infection. The cases reported here demonstrate the wide range of clinical presentations seen with PVL producing strains, which have so far been mainly associated with furuncles and necrotising pneumonia.

Juvenile thyrotoxicosis; can we do better?

Thyrotoxicosis remains a frustrating condition for the young person, family, and health professionals involved. The associated symptoms do not always suggest thyroid disease and patients can be unwell for many months before the diagnosis is made. The antithyroid drug regimen used to treat children and adolescents with thyrotoxicosis varies from one unit to another and yet the potentially life threatening side effects and remission rates post-treatment may be related to the regimen used. Most patients with thyrotoxicosis will need many years of drug therapy if the thyroid gland is not removed surgically or destroyed by radioiodine. Even "definitive" treatment will typically necessitate thyroxine replacement for life.

Cardiovascular malformations in infants of diabetic mothers.

OBJECTIVE: To compare the prevalence at live birth and the spectrum of cardiovascular malformations in infants born to diabetic mothers with pre-existing diabetes with that in infants of non-diabetic mothers. DESIGN: Prospective study of all live births in the resident population of one health region, with recording of details of the outcome of all pregnancies of women with pre-existing diabetes and of all live born babies with cardiovascular malformations. RESULTS: In the six years 1995-2000 there were 192 618 live births in the study population. Cardiovascular malformations were confirmed in 22 of 609 (3.6%) babies with diabetic mothers and in 1417 of 192 009 (0.74%) babies with non-diabetic mothers. The odds ratio for a cardiovascular malformation with maternal diabetes was 5.0 (95% confidence interval 3.3 to 7.8). Combination of these results with previous reports and comparison with the spectrum of cardiovascular malformations in infants of non-diabetic mothers shows a greater than threefold excess of transposition of the great arteries, truncus arteriosus, and tricuspid atresia. CONCLUSIONS: Pre-existing maternal diabetes is associated with a fivefold increase in risk of cardiovascular malformations. Transposition of the great arteries, truncus arteriosus, and tricuspid atresia are overrepresented to produce a substantial excess of these malformations.

Allosteric interactions within subsites of a monomeric enzyme: kinetics of fluorogenic substrates of PI-specific phospholipase C.

Two novel water-soluble fluorescein myo-inositol phosphate (FLIP) substrates, butyl-FLIP and methyl-FLIP, were used to examine the kinetics and subsite interactions of Bacillus cereus phosphatidylinositol-specific phospholipase C. Butyl-FLIP exhibited sigmoidal kinetics when initial rates are plotted versus substrate concentration. The data fit a Hill coefficient of 1.2-1.5, suggesting an allosteric interaction between two sites. Two substrate molecules bind to this enzyme, one at the active site and one at a subsite, causing an increase in activity. The kinetic behavior is mathematically similar to that of well-known cooperative multimeric enzymes even though this phosphatidylinositol-specific phospholipase C is a small, monomeric enzyme. The less hydrophobic substrate, methyl-FLIP, binds only to the active site and not the activator site, and thus exhibits standard hyperbolic kinetics. An analytical expression is presented that accounts for the kinetics of both substrates in the absence and presence of a nonsubstrate short-chain phospholipid, dihexanoylphosphatidylcholine. The fluorogenic substrates detect activation at much lower concentrations of dihexanoylphosphatidylcholine than previously reported.

Successful radioiodine treatment in a 3 year old child with Graves' disease following antithyroid medication induced neutropenia.

A 3 year old child with Graves' disease and mitral valve prolapse became neutropenic on carbimazole therapy. She was switched to propylthiouracil but the neutropenia recurred. She was treated with radioiodine but required two doses of 113 MBq and then 198 MBq five months later before becoming hypothyroid. The mitral valve prolapse resolved when she was euthyroid on thyroxine replacement. Antithyroid drugs, surgery, and radioiodine all have a place in the management of the thyrotoxic child.

Borjeson-Forssman-Lehmann syndrome and multiple pituitary hormone deficiency.

We describe two brothers with Borjeson-Forssman-Lehmann syndrome and the 22A-->T (Lys8X) PHF6 mutation, who presented with the symptoms and signs of multiple pituitary hormone deficiency. Biochemical investigations and radiology confirmed growth hormone (GH), thyroid stimulating hormone (TSH) and adrenocorticotrophic hormone (ACTH) as well as gonadotrophin deficiency. They were also found to have optic nerve hypoplasia. This family suggests that the BFL gene product may play an important role in midline neuro-development including the hypothalamo-pituitary axis.

Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line.

The primary objective of this study was to determine the sequence of biochemical signaling events that occur after modulation of the cellular redox state in the B cell lymphoma line, PW, with emphasis on the role of mitochondrial signaling. L-Buthionine sulphoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), was used to modulate the cellular redox status. The sequence and role of mitochondrial events and downstream apoptotic signals and mediators was studied. After BSO treatment, there was an early decline in cellular glutathione (GSH), followed by an increase in reactive oxygen species (ROS) production, which induced a variety of apoptotic signals (detectable at different time points) in the absence of any external apoptotic stimuli. The sequence of biochemical events accompanying apoptosis included a 95% decrease in total GSH and a partial (25%) preservation of mitochondrial GSH, without a significant increase in ROS production at 24h. Early activation and nuclear translocation of the nuclear factor kappa B subunit Rel A was observed at approximately 3h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24h of BSO treatment. p53 protein expression was unchanged after redox modulation for up to 72 h, and p21waf1 independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a time-dependent manner, beginning at 24h after BSO incubation. Irreversible commitment to apoptosis occurred between 48 and 72 h after BSO treatment when mitochondrial GSH was depleted, and there was an increase in ROS production. Procaspase 3 protein levels showed a time-dependent reduction following incubation with BSO, notably after 48 h, that corresponded with increasing ROS levels. At 96 h, caspase 3 cleavage products were detectable. The pan-caspase inhibitor zVADfmk, partially blocked the induction of apoptosis at 48 h, and was ineffective after 72 h. PW cells could be rescued from apoptosis by removing them from BSO after up to 48, but not 72 h incubation with BSO. Mitochondrial transmembrane potential (DeltaPsi(m)) remained intact in most of the cells during the 72 h observation period, indicating that DeltaPsi(m) dissipation is not an early signal for the induction of redox dependent apoptosis in PW cells. These data suggest that a decrease in GSH alone can act as a potent early activator of apoptotic signaling. Increased ROS production following mitochondrial GSH depletion, represents a crucial event, which irreversibly commits PW cells to apoptosis.

A genome-wide screen in Saccharomyces cerevisiae for genes affecting UV radiation sensitivity.

The recent completion of the deletion of essentially all of the ORFs in yeast is an important new resource for identifying the phenotypes of unknown genes. Each ORF is replaced with a cassette containing unique tag sequences that allow rapid parallel analysis of strains in a pool by using hybridization to a high-density oligonucleotide array. We examined the utility of this system to identify genes conferring resistance to UV irradiation by using a pool of 4,627 individual homozygous deletion strains (representing deletions of all nonessential genes). We identified most of the nonessential genes previously shown to be involved in nucleotide excision repair, in cell cycle checkpoints, in homologous recombination, and in postreplication repair after UV damage. We also identified and individually confirmed, by replacing the genes, three new genes, to our knowledge not previously reported to confer UV sensitivity when deleted. Two of these newly identified genes have human orthologs associated with cancer, demonstrating the potential of this system to uncover human genes affecting sensitivity to DNA-damaging agents and genes potentially involved in cancer formation.

Sensitive fluorescent quantitation of myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate by high-performance thin-layer chromatography.

A non-radioactive micro-assay for the cyclic phosphodiesterase reaction catalyzed by Bacillus cereus phosphatidylinositol-specific phospholipase C is described. The assay involves high-performance thin-layer chromatography on silica gel to resolve the substrate (myo-inositol 1,2-cyclic phosphate) and the product (myo-inositol 1-phosphate), followed by detection with a lead tetraacetate-fluorescein stain. The quantitation of these inositol phosphates in sample spots relative to a series of standards is accomplished by analysis of the fluorescent plate image with a commercial phosphoimager and associated software. The experimental considerations for reliable quantitation of inositol monophosphates in the range of 0.1 to 50 nmol are presented.

Synthesis of fluorogenic substrates for continuous assay of phosphatidylinositol-specific phospholipase C.

An improved synthesis of fluorogenic substrate analogues for phosphatidylinositol-specific phospholipase C (PI-PLC) is described. The water-soluble substrates, which are derived from fluorescein, are not fluorescent until cleaved by the enzyme, and provide a convenient means to continuously monitor PI-PLC activity. The improvement in the synthesis lies in the method used to protect the hydroxyl groups of the inositol portion of the substrate molecule and allows a milder deprotection procedure to be used. The result is a much more reproducible synthesis of the substrate. The improved procedure has been employed to synthesize a series of fluorogenic substrates, which differ in the length of the aliphatic tail attached to the fluorescein portion of the molecule. The length of the tail was found to have a significant effect on the rate of cleavage of these substrates.