3-Hydroxy-2-methylbutyryl-CoA dehydrogenase deficiency.

A boy now 8 years old presented at 21 months of age with developmental arrest, followed by regression, cortical blindness and myoclonic seizures. Urine organic acid analysis revealed 3-hydroxy-2-methylbutyric acid and tiglyglycine; 3-ketothiolase enzyme activity was normal and he was subsequently found to have 3-hydroxy-2-methylbutyryl-CoA dehydrogenase deficiency.

Males with epilepsy, complete subcortical band heterotopia, and somatic mosaicism for DCX.

Subcortical band heterotopia (SBH) is seen predominantly in females, resulting from mutations in the X-linked doublecortin (DCX) gene, and can present with mild mental retardation and epilepsy. Males carrying DCX mutations usually demonstrate lissencephaly and are clinically much more severely affected. This article reports two cases of males with SBH indistinguishable from the female phenotype, both resulting from somatic mosaicism for DCX mutation.

NudC associates with Lis1 and the dynein motor at the leading pole of neurons.

NUDC is a highly conserved protein important for nuclear migration and viability in Aspergillus nidulans. Mammalian NudC interacts with Lis1, a neuronal migration protein important during neocorticogenesis, suggesting a conserved mechanism of nuclear movement in A. nidulans and neuronal migration in the developing mammalian brain (S. M. Morris et al., 1998). To further investigate this possibility, we show for the first time that NudC, Lis1, and cytoplasmic dynein intermediate chain (CDIC) colocalize at the microtubule organizing center (MTOC) around the nucleus in a polarized manner facing the leading pole of cerebellar granule cells with a migratory morphology. In neurons with stationary morphology, NudC is distributed throughout the soma and colocalizes with CDIC and tubulin in neurites as well as at the MTOC. At the subcellular level, NudC, CDIC, and p150 dynactin colocalize to the interphase microtubule array and the MTOC in fibroblasts. The observed colocalization is confirmed biochemically by coimmunoprecipitation of NudC with CDIC and cytoplasmic dynein heavy chain (CDHC) from mouse brain extracts. Consistent with its expression in individual neurons, a high level of NudC is detected in regions of the embryonic neocortex undergoing extensive neurogenesis as well as neuronal migration. These data suggest a biochemical and functional interaction of NudC with Lis1 and the dynein motor complex during neuronal migration in vivo.

Cerebral gyral dysplasias: molecular genetics and cell biology.

The promise of genetics has been partly realized in our understanding of human brain development as this relates to disorders of gyral formation. Cerebral gyral dysplasias are disorders of brain formation that result in phenotypes with the common feature of abnormal cerebral gyri. This review emphasizes the recent progress made in understanding the human lissencephalies and related disorders. LIS1 heterozygous loss-of-function deletions and point mutations, as well as Doublecortin mutations in males, lead to a very similar phenotype, termed type 1 lissencephaly. Additionally, Doublecortin mutations in females lead to a more variable subcortical band heterotopia. Given the similarities between the lissencephaly phenotypes that result from aberrations in these genes, it is important to review the genetics of these disorders. In order to begin to understand the cell biology of the LIS1 protein and the Doublecortin protein, potentially interacting pathways need to be emphasized. Another human genetic disorder with an interestingly similar phenotype has a mouse correlate that has been well characterized. This surprising finding may lead to further understanding of LIS1 protein and of Doublecortin protein. Furthermore, mouse modeling of the aforementioned human disorders now holds promise for enabling us finally to understand the formation of the most complex organ that nature has produced - the human brain.

Neuronal platelet-activating factor receptor signal transduction involves a pertussis toxin-sensitive G-protein.

In most nonneural systems, platelet-activating factor (PAF) receptor effects are mediated by G-proteins that are often pertussis toxin-sensitive. The activation of pertussis toxin-sensitive G-proteins linked to PAF receptors results in the mobilization of intracellular calcium, at least in part, through the second messenger inositol triphosphate. We have sought to determine if a pertussis toxin-sensitive G-protein is involved in the PAF receptor-mediated phenomena of growth cone collapse and of synaptic enhancement in primary neuronal culture. Using infrared differential interference contrast microscopy and patch-clamp recording techniques, pertussis toxin, but not the inactive B oligomer of the toxin, was found to block both the growth cone collapse and the enhanced synaptic release of excitatory transmitter induced by a nonhydrolyzable PAF receptor agonist, making it likely that Go, Gq, or Gi is the G-protein transducer of PAF receptors in primary neurons. We believe that PAF acts directly on neuronal receptors, which are linked to pertussis toxin-sensitive G-proteins, on the tips of developing neurites, and on presynaptic nerve terminals, leading to growth cone collapse and enhanced synaptic release of transmitter.

Lissencephaly associated mutations suggest a requirement for the PAFAH1B heterotrimeric complex in brain development.

Human brain malformations, such as Miller-Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS) may result from abnormal neuronal migration during brain development. MDS and ILS patients have a hemizygous deletion or mutation in the LIS1 gene (PAFAH1B1), therefore, the LIS1 encoded protein (Lis1) may play a role in neuronal migration. Lis1 is a subunit of a brain platelet-activating factor acetylhydrolase (PAFAH1B) where it forms a heterotrimeric complex with two hydrolase subunits, referred to as 29 kDa (PAFAH1B3) and 30 kDa (PAFAH1B2). In order to determine whether this heterotrimer is required for the developmental functions of PAFAH1B, we examined the binding properties of 29 and 30 kDa subunits to mutant Lis1 proteins. The results defined the critical regions of Lis1 for PAFAH1B complex formation and demonstrated that all human LIS1 mutations examined resulted in abolished or reduced capacity of Lis1 to interact with the 29 and 30 kDa subunits, suggesting that the PAFAH1B complex participates in the process of neuronal migration.

Neuronal cytoskeletal alterations evoked by a platelet-activating factor (PAF) analogue.

Platelet-activating factor (PAF), a phospholipid signaling molecule found in brain, modulates several neural functions and is implicated in the human developmental brain disorder Miller-Dieker Lissencephaly (MDL). Exposure to PAF, and a non-hydrolyzable analogue, methyl carbamyl PAF (mc-PAF), produces the following rapid, reversible effects upon cultured hippocampal neurites: growth cone collapse, neurite retraction, and neurite varicosity formation. In this study, the cytoskeletal alterations that mediate these shape changes were investigated by comparing the effects of mc-PAF with other cytoskeletal-altering drugs, through the fluorescent labeling of cytoskeletal proteins and mitochondria, and by electron microscopy. Results indicate that rearrangements of microtubules (MTs), F-actin, and mitochondria underlie the neurite shape changes produced by mc-PAF. Evidence for MT alteration was obtained by comparing the effects of mc-PAF with nocodozole and taxol. Exposure to nocodazole, a MT-depolymerizing agent, produced growth cone collapse and neurite varicosity formation similar to mc-PAF, whereas pre-incubation of neurites in taxol, a MT-stabilizing drug, was effective in blocking mc-PAF-induced neurite effects. Immunofluorescent labeling and EM revealed MT splaying and unbundling within neurite varicosities following mc-PAF treatment. Immunofluorescent labeling also revealed that F-actin shifted from concentration in the growth cone to a diffuse distribution along the neurite shaft following mc-PAF exposure. Fluorescent labeling and EM also revealed retrograde movement and morphological alterations of mitochondria following mc-PAF exposure, resulting in mitochondrial aggregates within neurite varicosities. These cytoskeletal rearrangements may provide insights into the mechanisms by which PAF influences neuronal activity, and could have important implications for the impairment of neuronal motility observed in MDL.

Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality.

Heterozygous mutation or deletion of the beta subunit of platelet-activating factor acetylhydrolase (PAFAH1B1, also known as LIS1) in humans is associated with type I lissencephaly, a severe developmental brain disorder thought to result from abnormal neuronal migration. To further understand the function of PAFAH1B1, we produced three different mutant alleles in mouse Pafah1b1. Homozygous null mice die early in embryogenesis soon after implantation. Mice with one inactive allele display cortical, hippocampal and olfactory bulb disorganization resulting from delayed neuronal migration by a cell-autonomous neuronal pathway. Mice with further reduction of Pafah1b1 activity display more severe brain disorganization as well as cerebellar defects. Our results demonstrate an essential, dosage-sensitive neuronal-specific role for Pafah1b1 in neuronal migration throughout the brain, and an essential role in early embryonic development. The phenotypes observed are distinct from those of other mouse mutants with neuronal migration defects, suggesting that Pafah1b1 participates in a novel pathway for neuronal migration.

Platelet-activating factor receptor stimulation disrupts neuronal migration In vitro.

LIS-1 is a gene whose hemi-deletion causes the human neuronal migration disorder Miller-Dieker lissencephaly. It encodes a subunit of a brain platelet-activating factor (PAF) acetylhydrolase, an enzyme that inactivates PAF by hydrolyzing the acetyl moiety in the sn2 position of this phospholipid. Because PAF receptor activation has been shown to affect the developing neuronal cytoskeleton, we have hypothesized that a role for PAF in neurodevelopment is that of a modulator of neuroblast movement (a cytoskeletal function) and that an aberrant regulation of PAF could lead to an early arrest in migration. This report examines the effects of the nonhydrolyzable PAF receptor agonist methyl carbamyl PAF (mc-PAF) on the unidirectional in vitro migration of granule cells from cerebellar cell reaggregates on a laminin substrate. Bath treatment with mc-PAF yields a dose-dependent decrease in granule cell migration compared with controls. This effect can be blocked by the simultaneous bath application of BN 52021 and trans-BTD, PAF receptor-specific antagonists. Although mc-PAF minimally inhibited neurite growth, its primary effect was on somal movement along preextended neurites. These experiments suggest that the stimulation of neuronal PAF receptors could be one crucial step for the regulation of neuroblast migration and that disturbed PAF catabolism during neurodevelopment could contribute to the neuronal migration defects observed in Miller-Dieker lissencephaly.

Elvax as a slow-release delivery agent for a platelet-activating factor receptor agonist and antagonist.

The ability of the slow-release polymer, Elvax, to deliver a large number of different molecules to distinct brain regions has been well-documented. However, there are currently no reports of Elvax-mediated delivery of ether phospholipids, whose lipid structure renders them difficult to solubilize and detect under experimental conditions. In order to potentially examine the role of platelet-activating factor (PAF) receptors in the brain in vivo, we tested the ability of Elvax to release the ether phospholipid. PAF receptor agonist, methyl-carbamyl platelet-activating factor (mc-PAF), and the ginkolide, PAF receptor antagonist, BN 52021. Mc-PAF and BN 52021 containing Elvax sections (200 microns) prepared in methylene chloride were assayed for release characteristics with a rabbit platelet aggregation bioassay. We measured a slow, sustained release of mc-PAF and BN 52021 from Elvax in vitro over a 5 day period. Similar in vivo mc-PAF and BN 52021 release kinetics were determined. Therefore, we believe that this novel method of slow-release delivery of PAF receptor agonists and antagonists as measured with platelet aggregation is viable and offers a simple, sensitive and inexpensive method for potential in vivo studies of the roles of PAF and its receptors in neural systems.

Predominant localization of the LIS family of gene products to Cajal-Retzius cells and ventricular neuroepithelium in the developing human cortex.

Mutations that perturb neuronal migration provide important biological clues that can lead to an understanding of the role of specific cells and molecules in the formation of the cortex. The human neuronal migration disorder, Miller-Dieker Lissencephaly, results from a hemideletion of LIS-1, which encodes a subunit of a brain platelet-activating factor acetylhydrolase. The cellular localization of the LIS-1 gene product in human fetal brain and its normal role in neuronal migration have yet to be determined. LIS-1 belongs to a family of genes that have identical coding sequences (LIS-1 [chromosome 17] and LIS-2 [chromosome 2]). In the brain, LIS-1 is the more abundant gene as determined by Northern blot analysis. Using antibodies raised against 2 epitopes of the LIS-1/LIS-2 protein sequence, we have localized the LIS family of gene products in the developing human brain to the Cajal-Retzius cells, some subplate neurons, thalamic neurons, the ventricular neuroepithelium, and at later gestational ages, to the ependyma. Therefore, LIS-1 bears some resemblance to reelin, the gene product involved in the cortical mouse mutant reeler, in that Cajal-Retzius cells demonstrate immunolocalization. However, unlike reelin, LIS proteins are expressed not only in the Cajal Retzius cells, but also in the ventricular neuroepithelium, suggesting a potential role for this structure in neuronal migration.

Survey of sinonasal inverted papillomata for Epstein-Barr virus.

BACKGROUND: Several studies have indicated an etiologic role for viruses in the development of sinonasal inverted papillomata (IP). A recent report demonstrates a strong relationship (65%) between Epstein-Barr virus (EBV) and these lesions using polymerase chain reaction (PCR) analysis. METHODS: The present study analyzes a series of paraffin-embedded tissues, comprising 25 surgically resected IPs and four fungiform papillomata (FP) for the presence of EBV using a sensitive in situ hybridization (ISH) assay and PCR. RESULTS: None of the specimens examined showed evidence of EBV infection by ISH, and only two papillomata (one sinonasal IP and one FP) gave positive reactions for EBV using PCR. CONCLUSIONS: These data challenge the previous report and suggest that EBV is not a significant etiopathologic factor to be considered in the development of sinonasal IP.

Platelet-activating factor produces neuronal growth cone collapse.

It is generally believed that neuronal growth cone collapsing factors are proteins that interact with membrane-bound receptors. Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) - a phospholipid autocoid, also interacts with a membrane-bound neuronal receptor which is similar in nature to collapsing factor receptors. We report that PAF and the nonhydrolyzable PAF agonist, methyl carbamyl PAF (1-O-hexadecyl-2N-methylcarbamyl-sn-glycero-3-phosphocholine, mc-PAF), evoke a dose-dependent neuronal growth cone collapse. This collapse is specifically attenuated by the PAF receptor antagonist BN-52021. These data point to a PAF receptor-mediated growth cone collapse. Therefore, PAF must be added to the list of collapsing factors which potentially guide axons to their proper targets in the developing nervous system.

Analysis of urinary benzodiazepines using solid-phase extraction and gas chromatography-mass spectrometry.

A solid-phase extraction and GC-MS confirmation method was developed for certain urinary diazolo- and triazolobenzodiazepines, including the metabolites of lorazepam, clonazepam, alprazolam, and triazolam. The latter two do not form benzophenones, and the others are not readily confirmed by conventional thin-layer chromatography or GC-MS techniques. Samples were hydrolyzed with glucuronidase at 37 degrees C, adjusted to pH 4.5, extracted with Bond Elut Certify columns, dried, and derivatized using BSTFA with 1% TMCS. Sample preparation time averaged 4 hours. A GC-MS selected-ion-monitoring acquisition method targeting retention time, molecular ion abundances, and qualifier ion ratios was used to determine positive results. The recovery of 7-NH2-clonazepam was 95%, and recoveries of alpha-hydroxyalprazolam, alpha-hydroxytriazolam, and lorazepam were greater than 66%. Linearity was demonstrated from 0.1 to 1.0 microgram/mL for each drug. Within-run CVs were less than 11%, and between-run CVs were less than 16%. Using this technique, we have been able to confirm suspected cases of abuse that had not been confirmed by previous techniques.

Ethical dilemmas and decisions concerning the do-not-resuscitate patient undergoing anesthesia.

The growing geriatric population in this country makes it increasingly difficult to deal with the number of do-not-resuscitate (DNR) orders. In part, this is due to an increase in the number of elderly undergoing anesthesia and surgery. It can also be attributed to a rise in complex legal, ethical, and moral issues these orders pose for the healthcare professional caring for the DNR patients, including anesthetists. The term "DNR" is confusing to many, including healthcare professionals. As patients progress through the perioperative period, this confusion is compounded by the fact that administration of anesthesia encompasses interventions that include intubation, ventilation, and fluid replacement. These interventions may be regarded as resuscitative efforts outside the operating room. The anesthetist must identify and sort through a maze of conflicting courses of action, which must match the patient's desires and personal rights. The topic of DNR orders is addressed as well as some of the moral and ethical dilemmas they pose for the Certified Registered Nurse Anesthetist (CRNA). Some solutions are offered to help the nurse anesthetist make those decisions that are most "right" for the patient.

Platelet-activating factor as a potential retrograde messenger in CA1 hippocampal long-term potentiation.

Long-term potentiation (LTP) refers to a persisting enhancement of neurotransmission that follows high-frequency activation of certain synapses. Although both pre- and postsynaptic mechanisms contribute to LTP, it is believed that the enhanced release of neurotransmitter that accompanies this process results from the production of a diffusible messenger in postsynaptic neurons which traverses the synaptic cleft and alters the function of presynaptic terminals. One candidate for such a messenger is arachidonic acid, a metabolite produced by phospholipase A2 which augments synaptic transmission when coupled with presynaptic stimulation. However, the effects of arachidonic acid require activation of the postsynaptic receptor for N-methyl-D-aspartate. Previously we found that platelet-activating factor (1 O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), another phospholipase A2-derived messenger, selectively enhances excitatory postsynaptic currents in hippocampal neurons by a presynaptic mechanism. We now present evidence that platelet-activating factor, acting at a receptor localized to synaptic regions, participates in LTP in the CA1 region of rat hippocampal slices and may serve as part of a retrograde signalling cascade.