Influence of co-administration of oral insulin and docosahexaenoic acid in mice

Insulin and docosahexaenoic acid are both present in human milk. The aim of this study was to examine the effect of co-administration of oral insulin and DHA in mice. Immediately after weaning, Balb C mice were divided into four groups of seven mice each for a period of 4 weeks. Group 1 received a chow diet only. Group 2 received a chow diet and also was given human insulin (1 unit/mL of drinking water) without docosahexaenoic acid. Group 3 received a chow diet supplemented with docosahexaenoic acid (500 mg/kg/day in the chow) and no insulin. Group 4 received a chow diet and supplementation with both human insulin and docosahexaenoic acid. At 28 days, fasting blood levels of glucose, insulin, lipids, lipid peroxidation analysis, docosahexaenoic acid plasma levels, and docosahexaenoic acid content in red blood cells were determined. We found that glucose levels were lower in the group that was supplemented with insulin only (group 2, 61.4 mg/dL +/- 2.8,mean +/- SD) and in the group that was supplemented with DHA only (group 3, 61.1 mg/dL +/- 2.0) compared to controls (group 1, 71 mg/dL +/- 6.9, P < 0.0001). Supplementation of both insulin and docosahexaenoic acid (group 4) resulted in significantly lower glucose levels (56.4 mg/dL +/- 2.6) compared to those in groups 2 and 3 (P < 0.01). No significant differences were found in lipid profile or lipid peroxidation between the groups. We conclude that adding insulin or docosahexaenoic acid to the diet of weaned Balb C mice reduces glucose blood levels. Supplementation with both substances has a synergistic effect. The presence of insulin and docosahexaenoic acid in human milk may be the cause for reduced glucose levels in breast-fed infants, in addition to the known effects of DHA on insulin sensitivity.     

LIS1 missense mutations: variable phenotypes result from unpredictable alterations in biochemical and cellular properties

Mutations in one allele of the human LIS1 gene cause a severe brain malformation, lissencephaly. Although most LIS1 mutations involve deletions, several point mutations with a single amino acid alteration were described. Patients carrying these mutations reveal variable phenotypic manifestations. We have analyzed the functional importance of these point mutations by examining protein stability, folding, intracellular localization, and protein-protein interactions. Our data suggest that the mutated proteins were affected at different levels, and no single assay could be used to predict the lissencephaly phenotype. Most interesting are those mutant proteins that retain partial folding and interactions. In the case of LIS1 mutated in F31S, the cellular phenotype may be modified by overexpression of specific interacting proteins. Overexpression of the PAF-AH alpha1 subunit dissolved aggregates induced by this mutant protein and increased its half-life. Overexpression of NudE or NudEL localized this mutant protein to spindle poles and kinetochores but had no effect on protein stability. Our results implicate that there are probably different biochemical and cellular mechanisms obstructed in each patient yielding the varied lissencephaly phenotypes.     

Congenital dyserythropoietic anemia type I is caused by mutations in codanin-1

Congenital dyserythropoietic anemias (CDAs) constitute a rare group of inherited red-blood-cell disorders associated with dysplastic changes in late erythroid precursors. CDA type I (CDAI [MIM 224120], gene symbol CDAN1) is characterized by erythroid pathological features such as internuclear chromatin bridges, spongy heterochromatin, and invagination of the nuclear membrane, carrying cytoplasmic organelles into the nucleus. A cluster of 45 highly inbred Israeli Bedouin with CDAI enabled the mapping of the CDAN1 disease gene to a 2-Mb interval, now refined to 1.2 Mb, containing 15 candidate genes on human chromosome 15q15 (Tamary et al. 1998). After the characterization and exclusion of 13 of these genes, we identified the CDAN1 gene through 12 different mutations in 9 families with CDAI. This 28-exon gene, which is transcribed ubiquitously into 4738 nt mRNA, was reconstructed on the basis of gene prediction and homology searches. It encodes codanin-1, a putative o-glycosylated protein of 1,226 amino acids, with no obvious transmembrane domains. Codanin-1 has a 150-residue amino-terminal domain with sequence similarity to collagens and two shorter segments that show weak similarities to the microtubule-associated proteins, MAP1B (neuraxin) and synapsin. These findings, and the cellular phenotype, suggest that codanin-1 may be involved in nuclear envelope integrity, conceivably related to microtubule attachments. The specific mechanisms by which codanin-1 underlies normal erythropoiesis remain to be elucidated.     

Proteolysis of normal and mutated steroidogenic acute regulatory proteins in the mitochondria: the fate of unwanted proteins

Steroidogenic acute regulatory protein (StAR) is a nuclear encoded mitochondrial protein that enhances steroid synthesis by facilitating the transfer of cholesterol to the inner membranes of mitochondria in hormonally regulated steroidogenic cells. It is currently assumed that StAR activity commences before or during StAR import into the mitochondrial matrix. The present study was designed to demonstrate that, once imported and becoming physiologically irrelevant, exhaustive accumulation of StAR must be limited by a rapid degradation of the protein to prevent potential damage to the organelles. The use of uncouplers and manipulation of the interior mitochondrial pH in hormone-induced ovarian granulosa cells and StAR-expressing COS cells suggests that StAR degradation is biphasic and involves two classes of proteases. During phase I, which normally lasts for the first approximately 2 h following import, StAR is rapidly degraded by a protease, or proteases, that can be arrested by a nonclassical action of proteasome inhibitors such as MG132. StAR molecules that evade phase I are subjected to a second class of protease(s), which is slower and MG132 resistant. A third proteolytic entity was revealed in studies with C-28 StAR, a loss-of-function mutant of StAR. Upon initiation of its import, C-28 StAR dissipates the inner membrane potential and causes swelling of the mitochondria. Degradation of C-28 StAR, probably by an intermembrane space protease, is extremely rapid and MG132 insensitive. Collectively, this study defines StAR as the first naturally occurring mitochondrial protein that can serve as a substrate to probe multiple proteolytic activities in mammalian mitochondria.     

A model of MPTP-induced Parkinson's disease in the goldfish

Parkinson's disease (PD) has been modeled in humans, lower primates, and to a lesser extent in some other vertebrates by the administration of the potent neurotoxin 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP). The MPTP model has thus drawn considerable attention during the past 15 years, as a system to search for anti-PD drugs. It has been previously reported that a Parkinsonian syndrome can be elicited in the common goldfish (Carassius auratus) by a single dose of MPTP. This protocol describes the relatively simple and inexpensive MPTP model of PD in goldfish. The procedure takes 14-30 d, depending on how many animals are tested and on the planned study. The accessibility of the goldfish nervous system, neural density, the evolutionary equivalent subcortical circuitry and the greatly abbreviated blood-brain barrier of the goldfish brain, make it an attractive system for study of PD as well as potential drugs for therapy.     

High cooperativity of the SV40 major capsid protein VP1 in virus assembly

SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of approximately 6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility.     

Effects of a Dog-training Intervention on At-risk Youth

In the past few decades, interventions that involve animals have gained support among therapists, as they encourage verbal and non-verbal expressions of emotions. Our aim was to examine the impact of a dog-training intervention on the empathic skills of at-risk youth, compared with an intervention using empowerment groups. We hypothesized that participants in the dog-training group would report improved empathic abilities, compared with those in the comparison group. A prospective study was conducted, with a final sample of 55 at-risk youth: 30 in the dog-training group and 25 in the empowerment-based group (n = 25). Empathic skills were measured using the Interpersonal Reactivity Index, which consists of four subscales: fantasy abilities, perspective taking, empathic concern, and personal distress. We found a significant improvement in the empathic concern subscale only among participants in the dog-training group (F_(1,53) = 16.6, p < 0.01). This is discussed, along with limitations of the research and possible avenues for future research.     

Seemingly neutral polymorphic variants may confer immunity to splicing-inactivating mutations: a synonymous SNP in exon 5 of MCAD protects from deleterious mutations in a flanking exonic splicing enhancer

The idea that point mutations in exons may affect splicing is intriguing and adds an additional layer of complexity when evaluating their possible effects. Even in the best-studied examples, the molecular mechanisms are not fully understood. Here, we use patient cells, model minigenes, and in vitro assays to show that a missense mutation in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD) gene primarily causes exon skipping by inactivating a crucial exonic splicing enhancer (ESE), thus leading to loss of a functional protein and to MCAD deficiency. This ESE functions by antagonizing a juxtaposed exonic splicing silencer (ESS) and is necessary to define a suboptimal 3′ splice site. Remarkably, a synonymous polymorphic variation in MCAD exon 5 inactivates the ESS, and, although this has no effect on splicing by itself, it makes splicing immune to deleterious mutations in the ESE. Furthermore, the region of MCAD exon 5 that harbors these elements is nearly identical to the exon 7 region of the survival of motor neuron (SMN) genes that contains the deleterious silent mutation in SMN2, indicating a very similar and finely tuned interplay between regulatory elements in these two genes. Our findings illustrate a mechanism for dramatic context-dependent effects of single-nucleotide polymorphisms on gene-expression regulation and show that it is essential that potential deleterious effects of mutations on splicing be evaluated in the context of the relevant haplotype.