Real-Time PCR Quantitation of Clostridia in Feces of Autistic Children

Based on the hypothesis that intestinal clostridia play a role in late-onset autism, we have been characterizing clostridia from stools of autistic and control children. We applied the TaqMan real-time PCR procedure to detect and quantitate three Clostridium clusters and one Clostridium species, C. bolteae, in stool specimens. Group- and species-specific primers targeting the 16S rRNA genes were designed, and specificity of the primers was confirmed with DNA from related bacterial strains. In this procedure, a linear relationship exists between the threshold cycle (C_T) fluorescence value and the number of bacterial cells (CFU). The assay showed high sensitivity: as few as 2 cells of members of cluster I, 6 cells of cluster XI, 4 cells of cluster XIVab, and 0.6 cell of C. bolteae could be detected per PCR. Analysis of the real-time PCR data indicated that the cell count differences between autistic and control children for C. bolteae and the following Clostridium groups were statistically significant: mean counts of C. bolteae and clusters I and XI in autistic children were 46-fold (P = 0.01), 9.0-fold (P = 0.014), and 3.5-fold (P = 0.004) greater than those in control children, respectively, but not for cluster XIVab (2.6 × 10⁸ CFU/g in autistic children and 4.8 × 10⁸ CFU/g in controls; respectively). More subjects need to be studied. The assay is a rapid and reliable method, and it should have great potential for quantitation of other bacteria in the intestinal tract.     

Presence of a non-peptide morphine-like compound in human cerebrospinal fluid

A non-peptide morphine-like compound (MLC) is present in human ventricular and lumbar cerebro spinal fluid (CSF). None of the patients studied had received any narcotic drugs. MLC was determined by using a radioimmunoassay produced for morphine.     

The subcellular localization of phytanic acid oxidase in rat liver

Peroxisomal disorders (Zellweger's syndrome, neonatal adrenoleukodystrophy, infantile Refsum's syndrome, rhizomelic chondrodysplasia) show a series of enzymatic defects related to peroxisomal dysfunctions. Accumulation of phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) has been found in several of these patients, caused by a defect in the alpha-oxidation mechanism of this acid. The fact that the alpha-oxidation of phytanic acid is defective in the peroxisomal disorders as well as in classical Refsum's disease makes it likely that this oxidation normally takes place in the peroxisomes. A series of experiments preformed to localize the phytanic acid oxidase in subcellular fractions of rat liver show, however, that the alpha-oxidation of phytanic acid is a mitochondrial process. Free phytanic acid is the substrate, and the only cofactors necessary are ATP and Mg2+.     

Short-range linkage relationships of the valyl-tRNA synthetase gene in Fugu rubripes

 The class III region of the human major histocompatibility complex (MHC) is gene-dense, averaging one gene every 10–20 kilobases (kb). Its gene order has been compared with other organisms. To extend this analysis further in another non-mammalian vertebrate, the compact genome of Fugu rubripes was investigated for the existence of orthologues of these class III genes and their relative arrangements. Orthologues of the M _r 70000 heat shock protein (HSP70) and valyl-tRNA synthetase genes have been isolated. They do not seem to be closely physically linked as compared with mammals (supported by longer-range analysis using pulsed field gel electrophoresis). Random shotgun sequencing of the two Fugu cosmids containing the gene encoding valyl-tRNA synthetase revealed sequences resembling genes encoding tenascin-X, the nuclear antigen A/Ro of Sjogren’s syndrome, and the Landsteiner-Wiener blood group glycoprotein. These linkage relationships recapitulate some mammalian data, albeit imperfectly. Tenascin-X has been located both in the human and mouse Mhc class III regions. Three copies of a sequence found in the gene encoding Sjogren’s syndrome nuclear antigen A/Ro have been identified in the human Mhc class I region; the mouse Mhc class I region contains one copy. It is postulated that a fragmented gene pattern had existed prior to convergence in the ancestral mammalian immune response-related Mhc region, and that some of these genes had belonged to the same linkage group. Received: 17 February 1997 / Revised: 25 March 1997     

The ultrastructure of peripheral arteries during the development of DOCA hypertension in the rat

Summary The ultrastructure of the unconstricted superficial epigastric and femoral arteries is described in normal rats and in animals with hypertension induced by unilateral nephrectomy, by subcutaneous injections of desoxycorticosterone acetate and drinking of 1% NaCl. The femoral artery showed by far the greater response to the DOCA-saline treatment. In both vessels, the smooth-muscle cells changed from the normal spindle shape to a blunt ended outline with numerous pinocytotic vesicles and prolific collagen production. With long term hypertension, particularly in the femoral artery, the smooth-muscle cell profiles became very irregular. Hypertrophy of the organelles of the smooth-muscle cells was associated with an increase in the intercellular material which gradually changed from a mainly collagenous character to mainly vesicular. Lysosomal activity indicated cell disintegration. White blood cells adhere to the endothelium in hypertensive rats and there was an increase in subendothelial material. The number of intimal smooth-muscle cells increased noticeably in the femoral artery. In both arteries, the adventitial fibroblasts hypertrophied in hypertensive rats. In animals with an elevated blood pressure the morphological response was observed as early as 4 to 7 days after initiation of treatment.This work was supported by grants from the British Columbia Heart Foundation and the Medical Research Council of Canada.The authors are indebted to Miyoshi Nakashima for her invaluable assistance with these experiments.