Transgenic rat model of Huntington's disease

Huntington's disease (HD) is a late manifesting neurodegenerative disorder in humans caused by an expansion of a CAG trinucleotide repeat of more than 39 units in a gene of unknown function. Several mouse models have been reported which show rapid progression of a phenotype leading to death within 3-5 months (transgenic models) resembling the rare juvenile course of HD (Westphal variant) or which do not present with any symptoms (knock-in mice). Owing to the small size of the brain, mice are not suitable for repetitive in vivo imaging studies. Also, rapid progression of the disease in the transgenic models limits their usefulness for neurotransplantation. We therefore generated a rat model transgenic of HD, which carries a truncated huntingtin cDNA fragment with 51 CAG repeats under control of the native rat huntingtin promoter. This is the first transgenic rat model of a neurodegenerative disorder of the brain. These rats exhibit adult-onset neurological phenotypes with reduced anxiety, cognitive impairments, and slowly progressive motor dysfunction as well as typical histopathological alterations in the form of neuronal nuclear inclusions in the brain. As in HD patients, in vivo imaging demonstrates striatal shrinkage in magnetic resonance images and a reduced brain glucose metabolism in high-resolution fluor-deoxy-glucose positron emission tomography studies. This model allows longitudinal in vivo imaging studies and is therefore ideally suited for the evaluation of novel therapeutic approaches such as neurotransplantation.     

Heterogeneity in maple syrup urine disease: Aspects of cofactor requirement and complementation in cultured fibroblasts

Fibroblast strains derived from six patients with maple syrup urine disease have been investigated for their requirements of the cofactors NAD, CoASH, Mg⁺⁺ and TPP in comparison with 10 normal control strains. The reconstitution of the decarboxylase function of branched chain α-keto acid (BCKA) dehydrogenase complex in lysed cells was studied with respect to the substrates u-keto-isocaproic acid, α-keto-isovaleric acid, and α-keto-β-methylvaleric acid (KIC, KIVA, MEVA). The enzyme activity of all normal control strains for the substrates KIC and KIVA was not reconstituted by TPP + Mg⁺⁺ alone, but CoASH + NAD could reconstitute the enzyme activity with KIC and KIVA in different degrees. Only two control strains were tested with MEVA as substrate, and these showed in contrast that TPP + Mg⁺⁺ could partly reconstitute the enzyme activity. In contrast to the relative homogeneiy in the reconstitution profiles of normal strains, the five classical and one intermittent MSUD strains showed heterogeneity in cofactor requirements.
Complementation analysis using heterokaryons prepared from fibroblasts of four patients with classical MSUD and one patient with intermittent MSUD showed, in contrast to experiments with normal controls, a partial amelioration of the defect in two combinations; it is suggested that the defect in these strains is located at different functional subunits of the multienzyme complex.     

The effect of irradiation on the fever of delayed hypersensitivity

The effect of total body irradiation on the development of delayed hypersensitivity and on the febrile response to specific antigen has been studied in guinea-pigs with the following results:

1. 200 R. whole body irradiation in guinea-pigs, while suppressing circulating antibody response, did not prevent the development of delayed hypersensitivity.

2. Irradiated and non-irradiated hypersensitive animals had an equal febrile response to systemic challenge with specific antigen.

3. Serum from antigen-challenged, irradiated, hypersensitive animals contained a pyrogenic factor of the endogenous serum type capable of producing fever in normal recipients.

These results support the conclusion that production of circulating specific antibody is not essential either for development of delayed hypersensitivity or for the febrile response of the hypersensitive animal to specific antigen.

     

Studies on the mechanism of phorbol ester-induced inhibition of intercellular junctional communication

Intercellular gap-junctional communication was measured using[¹⁴C]citrulline incorporation in co-cultures of argininosuccinatelyase-deficient human fibroblasts and argininosuccinate synthetase-deficientChinese Hamster V79 cells. As previously shown, in this systemjunctional communication is completely inhibited by the tumorpromoting phorbol ester 12-O-tetra-decanoylphorbol-13-acetate(TPA). In the absence of extracellular calcium, TPA inhibitionwas less pronounced. However, synergism with calcium ionophoreA23187 could not be demonstrated. Chlorpromazine, trifluoperazineand 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl esterpartially antagonised the effect of TPA. No antagonism was demonstrablebetween calmidazolium and TPA. Treatment of co-cultures withexogenous phospholipase C or 1-oleoyl-2-acetylglycerol (OAG)resulted in communication inhibition, suggesting that proteinkinase C activation is involved in the mechanism of phorbolester-mediated communication inhibition. However co-cultureswhich had been made refractory to TPA by prolonged exposureto high concentrations remained sensitive to inhibition by phospholipaseC and OAG. These results suggest either that diacylglycerolcan produce other effects independent of protein kinase C activation,or that refractoriness to phorbol esters is not simply due toa decrease in the amount of protein kinase C.     

Precision Nutrition for Alzheimer’s Prevention in ApoE4 Carriers

The ApoE4 allele is the most well-studied genetic risk factor for Alzheimer’s disease, a condition that is increasing in prevalence and remains without a cure. Precision nutrition targeting metabolic pathways altered by ApoE4 provides a tool for the potential prevention of disease. However, no long-term human studies have been conducted to determine effective nutritional protocols for the prevention of Alzheimer’s disease in ApoE4 carriers. This may be because relatively little is yet known about the precise mechanisms by which the genetic variant confers an increased risk of dementia. Fortunately, recent research is beginning to shine a spotlight on these mechanisms. These new data open up the opportunity for speculation as to how carriers might ameliorate risk through lifestyle and nutrition. Herein, we review recent discoveries about how ApoE4 differentially impacts microglia and inflammatory pathways, astrocytes and lipid metabolism, pericytes and blood–brain barrier integrity, and insulin resistance and glucose metabolism. We use these data as a basis to speculate a precision nutrition approach for ApoE4 carriers, including a low-glycemic index diet with a ketogenic option, specific Mediterranean-style food choices, and a panel of seven nutritional supplements. Where possible, we integrate basic scientific mechanisms with human observational studies to create a more complete and convincing rationale for this precision nutrition approach. Until recent research discoveries can be translated into long-term human studies, a mechanism-informed practical clinical approach may be useful for clinicians and patients with ApoE4 to adopt a lifestyle and nutrition plan geared towards Alzheimer’s risk reduction.     

Trophic effect of angiotensin II in neonatal rat cardiomyocytes: role of endothelin-1 and non-myocyte cells

1. Angiotensin II (AII) and the endothelins (ET) are known to be potent trophic stimuli in various cells including cardiomyocytes. In order to characterize further these effects we studied, in neonatal rat ventricular cardiomyocytes, the effects of several endothelin-receptor antagonists and the AT₁-receptor antagonist losartan on AII- and endothelin-induced inositol phosphate (IP)-formation (assessed as accumulation of total [³H]-IPs in myo-[³H]-inositol prelabelled cells) and increase in rate of protein synthesis (assessed as [³H]-phenylalanine incorporation).
2. Endothelin (10 pM–1 μM) concentration-dependently increased IP-formation (max. increase at 100 nM ET-1: 130±14% above basal, n=25) and [³H]-phenylalanine incorporation (max. increase at 1 μM: 52±4% above basal, n=16) with an order of potency: ET-1>>ET-3. Both effects were antagonized by the ET_A/ET_B-receptor antagonist bosentan and the ET_A-receptor antagonist BQ-123, but not affected by the ET_B-receptor antagonist IRL 1038 and the AT₁-receptor antagonist losartan.
3. Pretreatment of the cells with 500 ng ml⁻¹ pertussis toxin (PTX) overnight that completely inactivated PTX-sensitive G-proteins did not attenuate but rather enhance ET-1-induced IP-formation. On the other hand, in PTX-pretreated cardiomyocytes ET-1-induced [³H]-phenylalanine incorporation was decreased by 39±5% (n=5).
4. AII (1 nM–1 μM) concentration-dependently increased IP-formation (max. increase at 1 μM: 42±7% above basal, n=16) and [³H]-phenylalanine incorporation (max. increase at 1 μM: 29±2%, n=9). These effects were antagonized by losartan, but they were also antagonized by bosentan and BQ-123.
5. In well-defined cultures of cardiomyocytes (not contaminated with non-myocyte cells) AII failed to increase [³H]-phenylalanine incorporation; addition of non-myocyte cells to the cardiomyocytes restored AII-induced increase in [³H]-phenylalanine incorporation.
6. We conclude that, in rat neonatal ventricular cardiomyocytes, (a) the ET-1-induced increase in rate of protein synthesis (through ET_A-receptor stimulation) involves at least two signalling pathways: one via a PTX-insensitive G-protein coupled to IP-formation, and the other one via a PTX-sensitive G-protein, and (b) the trophic effects of AII are brought about via local ET-1 secretion upon AT₁-receptor stimulation in neonatal rat ventricular non-myocyte cells.

     

Existential questions in early childhood programs in Sweden: Teachers' conceptions and children's experience

Swedish guidelines for early childhood education emphasize the importance of children's existential questions. The program encourages preschool to offer children different opportunities to work on questions like what it is to be, to live and die, grow old, about religion, beliefs, traditions, etc. at their level of maturity (Social-styrelsen, 1987). Our experience in teacher education and early childhood programs is, however, that this is an area in which many teachers have difficulties. In the present empirical study 13 teachers were interviewed to find outwhat they consider existential questions for their children to be, and whatstrategies or methods they have for dealing with these questions. Teachers' conceptions about the what and how aspects of existential questions are described in terms of their qualitative differences. Each teacher was then asked to keep a diary of the existential questions children raised during the following month. Differences between the teachers' conceptions, which showed hesitation, and the spontaneous approaches and active involvement that emerged in reality, as recorded in their own diaries, are discussed.     

In vitro effect of r-verapamil on acute myelogenous leukemia blast cells: studies of cytokine secretion and cytokine-dependent blast proliferation

The in vitro effect of the dextroisomer r-verapamil on blast cells derived from patients with acute myelogenous leukemia (AML) was studied. R-verapamil caused a dose-dependent inhibition of AML blast proliferation in the presence of stem-cell factor, leukemia inhibitory factor, interleukin 4, interleukin 6, and interleukin 10 when these cytokines were tested both alone and in different combinations. R-verapamil also inhibited the growth of clonogenic AML blast cells. The antiproliferative effect was not specific for AML blast cells, because r-verapamil also inhibited cytokine-dependent proliferation of blast cells derived from patients with acute lymphoblastic leukemia. The inhibitory effects of r-verapamil and anti-IL1 serum were additive, suggesting that the antiproliferative effect of r-verapamil does not depend solely on inhibition of IL1-mediated effects. Although r-verapamil inhibited spontaneous AML blast proliferation, for a majority of patients it caused only minimal, if any, inhibition of spontaneous cytokine secretion (IL1, IL1, TNF, IL6) by AML blast cells. Thus, although inhibition of IL1 effects may contribute in certain patients to the antiproliferative effect of r-verapamil, mechanisms other than IL1 inhibition seem to be more important in mediating the effects of r-verapamil.Abbreviations ALL Acute lymphocytic leukemia - AML acute myelogenous leukemia - cpm counts per minute - ELISA enzyme-linked immunosorbent assay - G-CSF granulocyte colony-stimulating factor - GM-CSF granulocyte-macrophage colony-stimulating factor - IL interleukin - IF leukemia inhibitory factor - PBMC peripheral blood mononuclear cells - RR relative response - SCF stem cell factor - TNF tumor necrosis factor