No abstract

To extract cell surface-associated proteins from living fungal cells, reducing agents such as β-mercaptoethanol and dithiothreitol are often used. We show here that both compounds are moderately lipophilic and may perturb the plasma membrane, thus causing the release of cytosolic proteins, especially at high extraction temperatures. To avoid artifacts, we recommend using (a) a low concentration of the reducing agent for only a short period of time, and (b) an extraction temperature of 4 • C to protect the integrity of the plasma membrane. Similarly, biotinylation of cell surface proteins should be carried out at low temperatures in the absence of dimethylsulphoxide.

One single, cytosolic aminopeptidase (AP N, EC 3.4.11.2) is found to be responsible for both leucyl-(leucylAP) and lysylaminopeptidase (lysylAP) activity detectable with whole cells of Lactococcus lactis subsp, cremoris strain HP. The existence of a cell-envelope-located form of this enzyme could be excluded. No restriction on the activity of the enzyme is imposed by the cell membrane if leucine-p-nitroanilide is used as the substrate; with lysine-p-nitroanilide the activity is highly cryptic. The enzyme has been purified and characterized. It is a metalloaminopeptidase with a molecular mass of 95 kDa. Co 2+ appears to be the most potent ion to (re)activate the enzyme; Zn 2+ and Mn 2+ are less effective. The AP N releases the positively charged amino acids and several uncharged (including proline) from the N-terminus. Ammonium salts affect the preference of the enzyme with respect to the N-terminal residue. A preferential interaction of the ammonium ion with an essential cation binding site seems to be responsible for the inhibition of lysylAP activity.

Inactivation of the gene encoding the 11‐kDa subunit VIII of yeast ubiquinol: cytochrome c oxidoreductase leads to an inactive complex, which lacks detectable cytochrome b [Maarse, A. C., De Haan, M., Schoppink, P. J., Berden, J. A. and Grivell, L. A. (1988) Eur. J. Biochem. 172, 179–184] and in which the steady‐state levels of the Fe‐S protein and the 14‐kDa subunit VII are severely reduced. When the 11‐kDa° mutant is transformed with a gene encoding a protein consisting of the 11‐kDa protein minus its last 11 amino acids and fused to a 7‐aminoacid sequence encoded by a stop oligonucleotide, the complex is assembled normally. Enzyme activity is similar to that of the wild type, as is also the sensitivity of the complex to antimycin and myxothiazol. Transformation of the mutant with a gene encoding a protein consisting of the 11‐kDa protein lacking the last 43 amino acids (i.e. almost half the protein) and fused to the same 7‐amino‐acid sequence as above, gives partial restoration of the complex. The Fe‐S protein and the 14‐kDa subunit VII still exhibit low steady‐state levels, but cytochrome b is present again, albeit at a strongly reduced level. Electron transport activity is also partially restored and correlates with the level of cytochrome b indicating that the turnover number of the complex is similar to that of wild‐type complex III. These findings demonstrate the important role played by the 11‐kDa protein in the stabilization of cytochrome b. They also imply that at least the C‐terminal half of the 11‐kDa protein is not part of an ubiquinol‐binding site. Moreover, since the deletion has no effect on the sensitivity of the complex to myxothiazol and antimycin, at least this part of the protein is probably not involved in binding of these inhibitors.

AIM The aim of this study was to improve the understanding of brain function in children with autism spectrum disorder (ASD) in relation to minor neurological dysfunctions (MNDs).METHOD We studied MNDs in 122 children (93 males, 29 females; mean age 8y 1mo, SD 2y 6mo) who, among a total cohort of 705 children (513 males, 192 females; mean age 9y, SD 2y 0.5mo) referred to a regional outpatient non-academic psychiatric centre in the Netherlands, were diagnosed with ASD after an extensive multidisciplinary psychiatric assessment. Children with clear neurological abnormalities (e.g. cerebral palsy or spina bifida) were excluded from the study. MNDs were assessed in all 705 children using the Touwen examination method. Special attention was paid to the severity and type of MND. Data of the children with ASD were compared with neurological morbidity data of children with other psychiatric disorders and with children in the general population, who were born at Groningen University Hospital between 1975 and 1978. RESULTS Seventy-four percent of the children with ASD showed complex MNDs compared with 52% of the children with other psychiatric disorders and 6% of the reference group (v 2 =18.0, p<0.001; v 2 =937.5, p<0.001 respectively). Specific dysfunctions frequently encountered in ASD were dysfunctional posture and muscle tone, fine manipulative disability, dyscoordination, and excessive associated movements.CONCLUSION These findings suggest a contribution of dysfunctional supraspinal networks involving multiple parts of the brain in the pathogenesis of ASD. This is consistent with findings from neuroimaging studies, and highlights the importance of neurological examinations in paediatric psychiatric assessments.Autism spectrum disorder (ASD) is an early childhood-onset neurodevelopmental disorder characterized by delay and deviations in the development of social, communicative, and cognitive skills, and is associated with a very wide range of syndrome expression. The term 'ASD' encompasses children with the full autism syndrome as well as variants, such as Asperger syndrome, and pervasive developmental disorders not otherwise specified.Children with ASD are often described as 'clumsy' 1-3 or as having motor deficits. [4][5][6] In addition, psychiatric classification systems cite stereotypes in motor behaviour (e.g. hand-flicking or -twisting) as criteria for classifying ASD. 7,8 However, little is known about the neurological background of the clumsiness. Some studies report that children with ASD frequently exhibit soft neurological signs, 9 but only rarely are details of these signs reported. Others report motor deficits but do not refer to the neurological background of these deficits. [4][5][6] Neuroimaging studies of ASD shed some light on the neural substrate of ASD. Abnormalities are described in the following neural systems: [10][11][12] (1) the cerebellum -loss of Purkinje cells and anomalies in the shape of the cerebellum and vermix; 13,14 (2) the cerebral cortex -hyperplasia of grey and white matter of ...