Normal sleep homeostasis and lack of epilepsy phenotype in GABA A receptor alpha3 subunit-knockout mice.

Thalamo-cortical networks generate specific patterns of oscillations during distinct vigilance states and epilepsy, well characterized by electroencephalography (EEG). Oscillations depend on recurrent synaptic loops, which are controlled by GABAergic transmission. In particular, GABA A receptors containing the alpha3 subunit are expressed predominantly in cortical layer VI and thalamic reticular nucleus (nRT) and regulate the activity and firing pattern of neurons in relay nuclei. Therefore, ablation of these receptors by gene targeting might profoundly affect thalamo-cortical oscillations. Here, we investigated the role of alpha3-GABA A receptors in regulating vigilance states and seizure activity by analyzing chronic EEG recordings in alpha3 subunit-knockout (alpha3-KO) mice. The presence of postsynaptic alpha3-GABA A receptors/gephyrin clusters in the nRT and GABA A-mediated synaptic currents in acute thalamic slices was also examined. EEG spectral analysis showed no difference between genotypes during non rapid-eye movement (NREM) sleep or at waking-NREM sleep transitions. EEG power in the spindle frequency range (10-15 Hz) was significantly lower at NREM-REM sleep transitions in mutant compared with wild-type mice. Enhancement of sleep pressure by 6 h sleep deprivation did not reveal any differences in the regulation of EEG activities between genotypes. Finally, the waking EEG showed a slightly larger power in the 11-13-Hz band in alpha3-KO mice. However, neither behavior nor the waking EEG showed alterations suggestive of absence seizures. Furthermore, alpha3-KO mice did not differ in seizure susceptibility in a model of temporal lobe epilepsy. Strikingly, despite the disruption of postsynaptic gephyrin clusters, whole-cell patch clamp recordings revealed intact inhibitory synaptic transmission in the nRT of alpha3-KO mice. These findings show that the lack of alpha3-GABA(A) receptors is extensively compensated for to preserve the integrity of thalamo-cortical function in physiological and pathophysiological situations.

Structure of a cephalosporin synthase.

Penicillins and cephalosporins are among the most widely used therapeutic agents. These antibiotics are produced from fermentation-derived materials as their chemical synthesis is not commercially viable. Unconventional steps in their biosynthesis are catalysed by Fe(II)-dependent oxidases/oxygenases; isopenicillin N synthase (IPNS) creates in one step the bicyclic nucleus of penicillins, and deacetoxycephalosporin C synthase (DAOCS) catalyses the expansion of the penicillin nucleus into the nucleus of cephalosporins. Both enzymes use dioxygen-derived ferryl intermediates in catalysis but, in contrast to IPNS, the ferryl form of DAOCS is produced by the oxidative splitting of a co-substrate, 2-oxoglutarate (alpha-ketoglutarate). This route of controlled ferryl formation and reaction is common to many mononuclear ferrous enzymes, which participate in a broader range of reactions than their well-characterized counterparts, the haem enzymes. Here we report the first crystal structure of a 2-oxoacid-dependent oxygenase. High-resolution structures for apo-DAOCS, the enzyme complexed with Fe(II), and with Fe(II) and 2-oxoglutarate, were obtained from merohedrally twinned crystals. Using a model based on these structures, we propose a mechanism for ferryl formation.

Severe malnutrition in children: high case-fatality rates can be reduced.

PIP: This article offers a protocol for reducing high case fatality rates from malnutrition. Most child deaths from malnutrition occur in the first few days of treatment. Treatment should involve stabilization followed by rehabilitation. The article describes the treatment procedures for hypoglycemia, hypothermia, dehydration, and missed infections and discusses feeding during the stabilization and rehabilitation phases of treatment. All severely malnourished children have excess body sodium but high intracellular and low plasma levels. Malnourished children have deficiencies of potassium and magnesium that may take 2 weeks to correct. Edema is partly due to deficiencies in potassium and magnesium. A high sodium intake can be corrected by rehydrating with a modified oral rehydration solution and the special starter formula. Family food should be prepared without salt. Magnesium and potassium should be added directly to foods. All severely malnourished children have vitamin and mineral deficiencies. Deficiencies may include vitamin A, zinc, copper, selenium, and folic acid. Multivitamin supplements can correct for micronutrient deficiencies. It is advised that zinc should not be ignored, since it is responsible for repair of intestinal mucosa, halting diarrhea, healing of ulcerated skin lesions, restoration of appetite, improved immune function, and lean tissue synthesis. Iron should not be given until growth starts, infections are controlled, and antioxidant status is improved (usually 1 week after admission). Early introduction of iron poses a risk of enhancing pathogen increases and stimulating production of toxic free radicals. Relapses can be reduced by training parents how to feed their child frequently with energy and nutrient dense foods. The regimen was tested in a South African project and found to reduce mortality from 30% to 20%. After greater hospital attention to treatment of sepsis and hypoglycemia, case fatality declined to 6%.^ieng

Why have mortality rates for severe malnutrition remained so high?

A review of the literature that has appeared over the past five decades indicates that the median case fatality from severe malnutrition has remained unchanged over this period and is typically 20-30%, with the highest levels (50-60%) being among those with oedematous malnutrition. A likely cause of this continuing high mortality is faulty case-management. A survey of treatment centres worldwide (n = 79) showed that for acutely ill children, inappropriate diets that are high in protein, energy and sodium and low in micronutrients are commonplace. Practices that could have fatal consequences, such as prescribing diuretics for oedema, were found to be widespread. Evidence of outmoded and conflicting teaching manuals also emerged. Since low mortality levels from malnutrition can be achieved using appropriate treatment regimens, updated treatment guidelines, which are practical and prescriptive rather than descriptive, need to be implemented as part of a comprehensive training programme.

Expression, purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from tomato in Escherichia coli.

1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyses the final step in the biosynthesis of the plant hormone ethylene. The successful overexpression and characterization of active ACC oxidase from tomato has been achieved. PCR was used to insert the corrected cDNA coding for the tomato ACC oxidase into the pET-11a expression vector. Cloning of the resultant construct in Escherichia coli BL21(DE3)pLysE gave transformants which expressed ACC oxidase at levels greater than 30% of soluble protein under optimized conditions. When induced by addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37 degrees C the ACC oxidase expressed was less soluble and less active than when induced at 27 degrees C. The enzyme was purified to near homogeneity by a three-step chromatographic procedure. The specific activity of the purified recombinant ACC oxidase was typically 1.3-1.9 mol of ethylene/mol of enzyme per min, higher than values reported for native enzyme. Like the native enzyme it displayed a requirement for ferrous iron and ascorbate, and CO2 was an activator. The ability to discriminate between racemic diastereomers of 1-amino-2-ethyl cyclopropane-1-carboxylic acid was demonstrated. The enzyme was found to have a loose specificity for ascorbate, showing apparent preference for D-ascorbate and 5,6-O-isopropylidene L-ascorbate rather than L-ascorbate. The addition of catalase, dithiothreitol and BSA to incubation mixtures all resulted in significant increases in activity. When treated with diethylpyrocarbonate (DEPC) under mildly acidic conditions, the enzyme rapidly lost activity. Comparison of the rate of inactivation with the increase in absorbance at 240 nm gave results consistent with the modification of two to three histidine residues at the active site, although the possibility of additional modification of other nucleophilic residues cannot be excluded. Inactivation was largely prevented by the addition of substrates and ferrous iron, implying that DEPC treatment results in the modification of active-site histidines, which act as ligands for ferrous iron. CO2 offered no protection against DEPC inactivation, either in the absence or presence of substrates and/or ferrous iron.

Gonococcal ophthalmia neonatorum despite treatment with antibacterial eye-drops.

Over a five-year period, 1964-8, 48 cases of gonococcal ophthalmia neonatorum were notified to the department of venereology in Glasgow. Thirty-seven babies were born in hospital and 11 at home. The conjunctivitis, usually recorded as a "sticky eye," developed between 1 and 13 days of birth, 36 by the fourth day.Diagnosis by culture of Neisseria gonorrhoeae was delayed in some cases up to 30 days after the appearance of the signs for those born in hospital and 15 days for those born at home, usually because of the blind use of antibacterial eye-drops which produced temporary alleviation of the signs without eradicating the infection; chloramphenicol was noteworthy in this respect.A "sticky eye" will resolve without the use of antibacterial agents, ophthalmia neonatorum will not. When it is decided to use an antibacterial agent pretreatment conjunctival smears for immediate staining and swabs for culture should be taken and the case notified to the medical officer of health.Gonococcal ophthalmia is a preventable disease. In view of the obstetricians' already heavily committed clinical work load there is need for venereologists to collaborate, on consultation and within the maternity hospitals wherever possible, in the screening of antenatal patients for candidiasis and trichomoniasis as well as for gonorrhoea. Some target groups, those with a pathological vaginal discharge or with certain adverse social factors, warrant more thorough investigation, while all those treated require further examination to ensure cure.