Epidermoid cysts associated with thoracic meningocele.

Intraspinal tumours of cutaneous origin associated with various spinal dysraphisms have been well documented in the literature. However, the metachronous development of intra- and extra-medullary tumours in conjunction with dorsal meningocele is rare. The authors report a patient with a thoracic dorsal meningocele and congenital intradural extramedullary epidermoid tumour. The patient developed an intramedullary epidermoid growth 12 years later. Subtotal resection of the tumour predisposed to a later recurrence. Meningocele is not always an isolated clinical entity but the concurrent occult lesions are usually veiled by the more conspicuous surface anomaly. Thorough magnetic resonance imaging of the whole neural axis helps to identify associated pathologies. Delicate intradural exploration by a microsurgical approach is necessary to achieve appropriate treatment.

Isolation of polyhydroxyalkanoates-producing bacteria using a combination of phenotypic and genotypic approach.

AIMS: To develop an efficient approach using a combination of phenotypic and genotypic methods for isolation of environmental bacteria that produce mid-chain-length polyhydroxyalkanoates (mcl-PHAs). METHODS AND RESULTS: A viable-colony staining method using Nile red was used to screen for PHA-producing bacteria followed by a polymerase chain reaction (PCR) screen using primers to amplify the partial nucleic acid sequence of the phaC1 synthase gene for confirmation. Microbes containing lipophilic storage compounds isolated from environmental samples could readily be detected by the colony staining method. They were further examined by Sudan Black staining to highlight the inclusions inside the cells. These isolates were subsequently subjected to PCR analysis. As a result, more than a hundred strains were identified as PHA-positive isolates from this screening approach. CONCLUSIONS: These results conclusively demonstrate that environmental bacterial strains able to accumulate the PHAs could readily be obtained by this screening method. SIGNIFICANCE AND IMPACT OF THE STUDY: We propose a polyphasic approach using a combination of phenotypic and genotypic screening method to rapidly screen and identify bacteria able to produce significant amounts of mcl-PHAs from environment. This approach can be adopted as a rapid screen for micro-organisms able to accumulate PHAs to be used for potential manufacture and other industrial applications.

Alteration of ischemic reperfusion injury in the rat neocortex by a potent antioxidant mexiletine.

The mechanisms by which mexiletine exerts its effects in increasing myocardial circulation, and smooth muscle perfusion and alleviating diabetic neuropathic pain have been widely discussed. The purpose of this study was to examine the protective effect of this compound in ischemia/reperfusion-induced cerebral injury following middle cerebral artery occlusion in Sprague-Dawley rats. Blood flow to the left cerebral hemisphere of the animals was interrupted by occluding the left cerebral artery and both carotid arteries simultaneously for 3 hrs. These animals were assigned to one of ten groups and divided into treatment group and pretreatment group; 1) control treatment group (n=8); 2) vehicle treatment group (n=8); 3) lower dose mexiletine (400 microg/kg) treatment group (n=8); 4) medium dose mexiletine (800 microg/kg) treatment group (n=8); 5) high dose mexiletine (2 mg/kg) treatment group (n=8); 6) control pretreatment group (n=8); 7) vehicle pretreatment group (n=8); 8) lower dose mexiletine (400 microg/kg) pretreatment group (n=8); 9) medium dose mexiletine (800 microg/kg) pretreatment group (n=8); and 10) high dose mexiletine (2 mg/kg) pretreatment group (n=8). The volume of cerebral infarction was measured in serial brain sections stained with triphenyltetrazolium chloride (TTC). Tissue infarction volume and tissue edema were estimated for each animal. The volume of cerebral infarction was significantly decreased in rats pretreated with mexiletine, and the ratio of tissue edema was also decreased as the dose of mexiletine increased. These results demonstrate that mexiletine, an anti-arrhythmic and use-dependent Na+ channel blocker, has protective effects in stroke at concentrations sufficient to confer significant protection, as measured by the volume of infarction and brain edema index in a model of focal, neocortical ischemia in Sprague-Dawley rats.

Q/R RNA editing of the AMPA receptor subunit 2 (GRIA2) transcript evolves no later than the appearance of cartilaginous fishes.

The amino acid, either a glutamine (Q) or an arginine (R), at the Q/R site of the pore-lining segment (M2) of a vertebrate AMPA receptor subunit critically influences the properties of the receptor. The R codon of the mammalian AMPA receptor subunit 2 (GRIA2) transcript is not coded by the chromosomal sequence, but is created by posttranscriptional RNA editing activities. On the other hand, the R codons of some teleost GRIA2 homologs are coded by chromosomal sequences. To elucidate the evolution of the utilization of Q/R RNA editing in modifying vertebrate GRIA2 transcripts, the GRIA2 genes of five fish species and an amphibian were studied. The putative hagfish GRIA2 homolog (hfGRIA2) encodes an R codon, whereas shark and bullfrog GRIA2 genes specify a Q codon at the genomic Q/R site. All gnathostoma GRIA2 genes possess an intron splitting the coding regions of M2 and the third hydrophobic region (M3). The intronic components required for Q/R RNA editing are preserved in all the Q-coding vertebrate GRIA2 genes but are absent from the R-coding GRIA2 genes. Interestingly, the hfGRIA2 is intronless, suggesting that hfGRIA2 is unlikely evolved from a Q/R editing-competent gene. Results of this study suggest that modification of GRIA2 transcripts by Q/R editing is most likely acquired after the separation of the Agnatha and Gnathostome.

Identifications, classification, and evolution of the vertebrate alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit genes.

The AMPA receptor (AMPAR), a pharmacologically defined ionotropic glutamate receptor, mediates fast excitatory synaptic transmission in the vertebrate central nervous system. Mammalian and avian AMPARs are assembled from the products of four genes (GRIA1-GRIA4) conserved in their translated sequences and gene organizations. Teleost fish also express AMPAR subunits; however, the AMPAR genes have not been extensively investigated in lower vertebrates. To elucidate the evolution of vertebrate AMPAR genes, reverse-transcriptase PCR-based surveys of subunits expressed in the brains of eight nonmammalian vertebrates were performed. The newly cloned vertebrate AMPAR subunits were classified by their sequence identities to the mammalian AMPAR subunits. The results of molecular and phylogenetic analyses indicated that the members of the AMPAR gene family increased from two in the jawless hagfish to four in the tetrapods and the shark and to more than four in the teleost fish. The sizes of AMPAR gene families correlate well with those of many multigene families observed in various vertebrates. Moreover, all vertebrates expressed at least one AMPAR subunit bearing an arginine (R) at the Q/R site, at which no invertebrate glutamate receptor subunit has been found to have an R residue, suggesting that the low calcium-permeable AMPARs appeared at early evolutionary stages of vertebrate central nervous systems. Uniquely, the loop 1 (L1) regions between hydrophobic domain 1 and hydrophobic domain 2 of the hagfish putative GRIA2 and all the teleost GRIA1 subunits were much longer than those of the remaining known ionotropic glutamate receptor subunits. The length and sequence of the L1 of teleost GRIA1 subunits were heterogeneous, suggesting that the amino acid residues in L1 were not highly selected.

Traumatic lumbar spinal subdural hematoma--a case report.

A 74-year-old woman suffered from lower legs weakness after a motor vehicle accident. Magnetic resonance imaging (MRI) on the seventh posttraumatic day, revealed a lumbar spinal subdural hematoma at the level of L4-5. After surgical intervention to remove the subdural hematoma, the patient made a complete recovery.

Genomic organization of the Oreochromis mossambicus glutamate receptor subunit 2 beta gene (fGluR2 beta): presence of two different 5'-untranslated regions.

The AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid)-preferring receptor is one of the pharmacologically defined ionotropic glutamate receptors, which mediate fast excitatory synaptic transmission in the central nervous system of vertebrates. Here, we report the mapping of the transcriptional start points and identification of the intron-exon boundaries of the teleost AMPA receptor subunit gene fGluR2 beta. fGluR2 beta and the mouse GluR2 share a similar genomic organization, having identical intron insertion sites and a large intron 2; however, fGluR2 beta has an extra exon encoding an alternate 5'-UTR. Results of RT-PCR and RNase protection analyses indicate that mature fish brain expresses two types of fGluR2 beta transcripts with different 5' ends. Transcriptions of these two fGluR2 beta transcripts started from two chromosomal regions separated by at least 10 kb. Only the transcript starting from the region more upstream on the chromosome was spliced. Moreover, transcript initiated from the downstream region was more abundant than that initiated from the upstream region.

Molecular analysis of cDNA molecules encoding glutamate receptor subunits, fGluR1 alpha and fGluR1 beta, of Oreochromis sp.

In this study, we isolated two cDNA molecules encoding putative glutamate receptor subunits, fGluR1 alpha and fGluR1 beta, from an Oreochromis sp. brain cDNA library by hybridizing with the glutamate receptor cDNA, fGluR2 beta, of the same fish. The deduced amino acid sequence of the fGluR1 alpha consists of 908 residues with an 18-residue signal peptide and displays a sequence identity of 74% to the amino acid sequence of rat GluR1 subunit. Northern blotting indicates that the expression level of fGluR1 alpha in telencephalon is higher than that in optic tectum and cerebellum in adult fish brain. Reverse-transcriptase polymerase chain reaction and genomic analyses reveal the presence of variants created by alternative splicing at the flip-flop module and the carboxyl terminus of fGluR1 alpha transcripts. The amino acid sequence of fGluR1 alpha is unique in that it contains a glutamine-rich sequence inserted at the loop 1 (L1) between transmembrane domains 1 and 2. A second incomplete cDNA clone, designated fGluR1 beta, coding for a polypeptide showing sequence identity to the rat GluR1 and fGluR1 alpha was isolated from the same library. Insertion of a serine- and glutamine-rich sequence at the L1 was also detected in the translated sequence of fGluR1 beta.

Determination of relative abundance of splicing variants of Oreochromis glutamate receptors by quantitative reverse-transcriptase PCR.

In this study, the relative abundance of splicing variants of Oreochromis non-NMDA subtype glutamate receptors was studied by quantitative reverse-transcriptase PCR (RT-PCR). The relative expression level between the flip and flop transcripts of fGluR2 alpha determined by quantitative RT-PCR is apparently much higher than that estimated by sequence analysis of the cloned RT-PCR products. Control studies were performed to demonstrate the accuracy of the application of quantitative RT-PCR analysis in studying the relative abundance between the flip and flop transcripts of glutamate receptors.

Characterization of two fish glutamate receptor cDNA molecules: absence of RNA editing at the Q/R site.

Two cDNA clones encoding putative ionotropic glutamate receptor subunits were isolated from a brain cDNA library of a freshwater fish, Oreochromis sp. The deduced amino acid sequences of these two cDNAs, fGluR2 alpha and fGluR2 beta, display the highest sequence identity (85%) to that of the rat GluR2 (AMPA receptor subunit) and they contain an arginine codon at the Q/R editing site of the TM2 segment. Genomic sequence analysis of the exons encoding the TM2 reveals the presence of an arginine codon at the Q/R site, suggesting that the RNA editing mechanism acting in the mammalian GluR2 does not operate at the homologous site in these two fish genes. In contrast to the absence of RNA editing at the Q/R site, transcripts of fGluR2 alpha and fGluR2 beta are subjected to RNA editing at a second site, the R/G site. A splicing variant of fGluR2 alpha, fGluR2 alpha-c, with a shorter C-terminal sequence was found; however, no C-terminal splicing variant of fGluR2 beta was detected in the mature fish. Similar to the mammalian AMPA receptor, variants created by the alternate choice of flip and flop modules were found among transcripts of fGluR2 alpha-c and fGluR2 beta. The amino acid sequences of flip and flop modules of fGluR2 beta are identical to that of the rat GluR2, whereas the amino acid sequences of the flip and flop modules of fGluR2 alpha-c differ from the invariant consensus sequences of the rat AMPA receptor subunits.

Molecular characterization of a deletion-prone region of plasmid pAE1 of Alcaligenes eutrophus H1.

A 93-kb region (D region) of plasmid pAE1 of Alcaligenes eutrophus H1 has been found to have a high rate of spontaneous deletion. In this study, we constructed a restriction endonuclease map and carried out limited sequencing of an approximately 100-kb region from pAE1 which includes the D region (the deleted region) in order to detect and characterize repetitive sequences. Two types of repetitive sequences, the R1 and R2 sequences, were observed to flank the D region; within the D region are three copies of insertion element ISAE1. The R1 and R2 sequences are arranged in direct and inverted orientations, respectively. Molecular analysis of the end product of the deletion is consistent with the hypothesis that the loss of the D-region DNA is the result of recombination between two copies of the R1 sequence. The R1 sequence encodes a 415-amino-acid protein which exhibits substantial sequence similarity to the lambda integrase family of site-specific recombinases. Its genetic function remains to be determined.

Molecular and genetic characterization of an Alcaligenes eutrophus insertion element.

An insertion element, ISAE1, was discovered during the molecular analysis of mutants defective in the autotrophic growth (Aut-) of Alcaligenes eutrophus H1-4, a mitomycin C-generated derivative of strain H1. ISAE1 is 1,313 bp long, has 12-bp nearly perfect inverted terminal repeats, and contains an open reading frame that has a coding capacity of 408 amino acids. Direct repeats of 8 bp were generated by insertion of ISAE1 into chromosomes or plasmids. Most insertion were found in the AT-rich target sites. The distribution of ISAE1 is limited to A. eutrophus H1 (ATCC 17698) and H16 (ATCC 17699). Variants with newly transposed copies of ISAE1 could be isolated at an elevated frequency by changing the growth conditions.

N-acetyl-D-glucosamine-asparagine structure in ribosome-inactivating proteins from the seeds of Luffa cylindrica and Phytolacca americana.

Glycosylation-site-containing peptides were isolated from the proteolytic digests of luffin-a, luffin-b, PAP-S and CNBr-fragments of PAP-S by reverse-phase HPLC, and their amino acid compositions and sequences were analyzed. Six peptides were obtained from luffin-a, and three each from luffin-b and PAP-S. All of these peptides were negative toward the phenol-H2SO4 reaction and gave only N-acetyl-D-glucosamine in gas chromatography after methanolysis and reacetylation. Amounts of N-acetyl-D-glucosamine in these peptides were determined as D-glucosamine to be approximately one mol per peptide by an amino acid analyzer after HCl hydrolysis. Based on these results we concluded that Asn residues at positions of 28, 33, 77, 84, 206, and 227 in luffin-a, of 2, 78, and 85 in luffin-b, and of 10, 44, and 255 in PAP-S were glycosylated with only GlcNAc, and contained one residue per site.

The complete amino acid sequence of antiviral protein from the seeds of pokeweed (Phytolacca americana).

The complete amino acid sequence of antiviral protein from the seeds of pokeweed (Phytolacca americana) has been determined. This has been done by the sequence analysis of peptides derived by enzymatic digestion with trypsin, pepsin, and lysylendopeptidase, as well as by chemical cleavage with cyanogen bromide. The protein consists of 261 amino acid residues containing two disulfide bonds and has a calculated molecular mass of 29167 Da. The two disulfide bonds connect Cys-34 to Cys-258 and Cys-84 to Cys-105. Comparison of this sequence with the sequence of the ricin A-chain shows that there are identical residues at 76 positions in the two molecules (30% identity), having an extended region of highly conserved sequence at positions 170-183 (IQMXSEAARFXYIE). In contrast, the internal regions at positions 77-119 and 141-169 and the C-terminal 15 amino acid residues are less homologous in the two proteins.