Visuospatial processing and the function of prefrontal-parietal networks in autism spectrum disorders: a functional MRI study

Objective: Individuals with autism spectrum disorders typically have normal visuospatial abilities but impaired executive functioning, particularly in abilities related to working memory and attention. The aim of this study was to elucidate the functioning of frontoparietal networks underlying spatial working memory processes during mental rotation in persons with autism spectrum disorders. Method: Seven adolescent males with normal IQ with an autism spectrum disorder and nine age- and IQ-matched male comparison subjects underwent functional magnetic resonance imaging scans while performing a mental rotation task. Results: The autism spectrum disorders group showed less activation in lateral and medial premotor cortex, dorsolateral prefrontal cortex, anterior cingulate gyrus, and caudate nucleus. Conclusions: The finding of less activation in prefrontal regions but not in parietal regions supports a model of dysfunction of frontostriatal networks in autism spectrum disorders.

Processing of mutated human proinsulin to mature insulin in the non-endocrine cell line, CHO

Heterologous genes encoding proproteins, including proinsulin, generally produce mature protein when expressed in endocrine cells while unprocessed or partially processed protein is produced in non-endocrine cells. Proproteins, which are normally processed in the regulated pathway restricted to endocrine cells, do not always contain the recognition sequence for cleavage by furin, the endoprotease specific to the constitutive pathway, the principal protein processing pathway in non-endocrine cells. Human proinsulin consists of B-Chain-C-peptide-A-Chain and cleavage at the B/C and C/A junctions is required for processing. The B/C, but not the C/A junction, is recognised and cleaved in the constitutive pathway. We expressed a human proinsulin and a mutated proinsulin gene with an engineered furin recognition sequence at the C/A junction and compared the processing efficiency of the mutant and native proinsulin in Chinese Hamster Ovary cells. The processing efficiency of the mutant proinsulin was 56% relative to 0.7% for native proinsulin. However, despite similar levels of mRNA being expressed in both cell lines, the absolute levels of immunoreactive insulin, normalized against mRNA levels, were 18-fold lower in the mutant proinsulin-expressing cells. As a result, there was only a marginal increase in absolute levels of insulin produced by these cells. This unexpected finding may result from preferential degradation of insulin in non-endocrine cells which lack the protection offered by the secretory granules found in endocrine cells.

Super-CHO - A cell line capable of autocrine growth under fully defined protein-free conditions

Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/10(6)cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/10(6)cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/10(6)cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media.

Centrifugal processing of cell debris and inclusion bodies from recombinant Escherichia coli

The settling characteristics of cell debris and inclusion bodies prior to, and following, fractionation in a disc-stack centrifuge were measured using Cumulative Sedimentation Analysis (CSA) and Centrifugal Disc photosedimentation (CDS). The impact of centrifuge feedrate and repeated homogenisation on both cell debris and inclusion body collection efficiency was investigated. Increasing the normalised centrifuge feedrate (Q/Sigma) from 1.32 x 10(-9) m s(-1) to 3.97 x 10(-9) m s(-1) leads to a 36% increase in inclusion body paste purity. Purity may also be improved by repeated homogenisation. Increasing the number of homogeniser passes results in smaller cell debris size whilst leaves inclusion body size unaltered. At a normalised centrifuge feedrate of 2.65 x 10(-9) m s(-1), increasing the number of homogeniser passes from two (2) to ten (10) improved overall inclusion body paste purity by 58%. Grade-efficiency curves for both the cell debris and inclusion bodies have also been generated in this study. The data are described using an equation developed by Mannweiler (1989) with parameters of k = 0.15-0.26 and n = 2.5-2.6 for inclusion bodies, and k = 0.12-0.14 and n = 2.0-2.2 for cell debris. This is the first accurate experimentally-determined grade efficiency curve for cell debris. Previous studies have simply estimated debris grade efficiency curves using an approximate debris size distribution and grade efficiency curves determined with 'ideal particles' (e.g. spherical PVA particles). The findings of this study may be used to simulate and optimise the centrifugal fractionation of inclusion bodies from cell debris.

Mild head injury and speed of information processing: A prospective study of professional rugby league players

The sensitivity of several short tests of speed of information processing to the effects of mild head injury in rugby league football was investigated. The measures used were the Symbol Digit Modalities Test, the Digit Symbol Substitution Test, and the Speed of Comprehension Test. Two studies were conducted, the first to examine the effect of practice, the second to determine sensitivity to cognitive impairment immediately following injury. The first study established alternate form equivalence and demonstrated that performance on the Speed of Comprehension and Digit Symbol Substitution tests improved with practice, whereas the Symbol Digit Modalities test remained stable. A second study of 10 players who subsequently sustained mild head injuries showed that measures of speed of information processing were sensitive to impairment in the postacute phase, whereas an untimed task of word recognition (Spot-the-Word) was not. Speed of Comprehension was more sensitive to postinjury impairment than either the Digit Symbol Substitution or Symbol Digit Modalities tests. A repeated baseline assessment before injury using the higher score to reflect a player's potential, allowed measurement of impaired performance on sensitive tests.

The role of xenobiotic metabolizing enzymes in arylamine toxicity and carcinogenesis: Functional and localization studies

In both animal models and humans, the first and obligatory step in the activation of arylamines is N-hydroxylation. This pathway is primarily mediated by the phase-I enzymes CYP1A1, CYP1A2 and CYP4B1. In the presence of flavonoids such as alpha-naphthoflavone and flavone, both CYP3A4 and CYP3A5 have also been shown to play a minor role in the activation of food-derived heterocyclic amines. The further activation of N-hydroxyarylamines by phase-II metabolism can involve both N,O-acetylation and N,O-sulfonation catalyzed by N-acetyltransferases (NAT1 and NAT2) and sulfotransferases, respectively. Using an array of techniques, we have been unable to detect constitutive CYP1A expression in any segments of the human gastrointestinal tract. This is in contrast to the rabbit where CYP1A1 protein was readily detectable on immunoblots in microsomes prepared from the small intestine. In humans, CYP3A3/3A4 expression was detectable in the esophagus and all segments of the small intestine. Northern blot analysis of eleven human colons showed considerable heterogeneity in CYP3A mRNA between individuals, with the presence of two mRNA species in same subjects. Employing the technique of hybridization histochemistry (also known as in situ hybridization), CYP4B1 expression was observed in some human colons but not in the liver or the small intestine. Hybridization histochemistry studies have also demonstrated variable NAT1 and NAT2 expression in the human gastrointestinal tract. NAT1 and NAT2 mRNA expression was detected in the human liver, small intestine, colon, esophagus, bladder, ureter, stomach and lung. Using a general aryl sulfotransferase riboprobe (HAST1), we have demonstrated marked sulfotransferase expression in the human colon, small intestine, lung, stomach and liver. These studies demonstrate that considerable variability exists in the expression of enzymes involved in the activation of aromatic amines in human tissues. The significance of these results in relation to a role for heterocyclic amines in colon cancer is discussed.

Enhancing the phonological processing skills of children with specific reading disability

The present study evaluated the benefits of phonological processing skills training for children with persistent reading difficulties. Children aged between 9-14 years, identified as having a specific reading disability, participated in the study. In a series of three experiments, pedagogical issues related to length of training time, model of intervention and severity of readers' phonological processing skills deficit prior to intervention, were explored. The results indicated that improvement in poor readers' phonological processing skills led to a dramatic improvement in their reading accuracy and reading comprehension performance. Increasing the length of training time significantly improved transfer effects to the reading process. Children with particularly severe phonological processing skill deficits benefited from art extended training period, and both individual and group intervention models for phonological processing training proved successful. Implications for speech and language therapists are discussed.