Differential expression of LMO4 protein in Alzheimer's disease.

The molecular bases of late-onset and sporadic Alzheimer's disease (AD) still have to be unraveled. Among putative candidates for molecular variations in AD, we propose LMO4 protein, a transcription regulator, involved in multiple protein complexes. We investigated changes in LMO4 immunoreactivity in vulnerable brain regions of AD cases and controls of comparable age. Immunocytochemical analysis revealed a high level of LMO4 expression in the entorhinal cortex (EC) and in the CA1 hippocampal region of the control brains and a consistent decrease in the AD brains, correlated with the amount of neurofibrillary tangles (NFT) degenerating neurones and the severity of senile plaques deposition. The decrease in LMO4 immunoreactivity resulted both from weaker immunoreactive signals and from a loss of immunoreactive neurones. LMO4 immunocytochemical staining appeared not to be colocalized with NFT in a majority of neurones. Its expression was weak in the dentate gyrus and stronger in CA3-4, two regions with no or low numbers of NFT, but there was no decrease in AD compared to control cases. In the frontal cortex, the ventro-infero-median region (area 12) showed a greater LMO4 expression than the polar one (area 9), but no decrease in AD was observed. As LMO4 has been proposed to inhibit cellular differentiation, it can be hypothesized that a reduced expression is associated in EC and CA1 with attempts of diseased neurones to differentiate (e.g. compensatory neuritogenesis). Taken together, these data indicate that LMO4 protein is involved in the complexity of the disease phenotype, at least as a secondary factor.

Sexing chick embryos: a rapid and simple protocol.

1. Analysis of gene expression in the developing chick gonads requires the collection of male and female tissues from embryos between 3.5 d and 8.5 d of development. However, male and female chick embryos are indistinguishable by morphological examination before d 7.5 of development. 2. Sex identification of earlier embryos is only possible by molecular methods, which at present are laborious and time consuming. 3. We have devised a PCR-based sexing protocol which combines both sex specific and control reactions in a single tube assay. The assay is rapid and effective over a wide range of DNA concentrations and is tolerant of poor quality DNA. 4. Procedures are described for identifying the sex of individual embryos using either tissue samples or a small number of cells recovered from amniotic fluid.

Altered apolipoprotein D expression in the brain of patients with Alzheimer disease.

The etiology of late-onset Alzheimer disease is poorly understood. Predisposing factors such as the apolipoprotein E4 allele, as well as protective factors (e.g., antioxidants) have been proposed to play a role in the disease's process. A search for predisposing factors contributing to sporadic late-onset Alzheimer disease was initiated using the differential display technique. RNA expression profiles of the entorhinal cortex and the cerebellum of Alzheimer-diseased and normal patients were compared. The entorhinal cortex is the first brain region to accumulate neurofibrillary tangles during disease progression, whereas the cerebellum is spared. In the Alzheimer cases of this study, one signal showing preferential expression in the entorhinal cortex corresponded to the apolipoprotein D gene. This preferential expression might be genuine at the RNA level as suggested by the in situ hybridization method used. In addition, immunohistochemical experiments showed higher percentages of Apolipoprotein D reactive pyramidal neurons in the entorhinal cortex and region 1 of Ammon's horn in diseased patients. This increase correlated with the number of neurofibrillary tangles in Alzheimer as well as in normal patients. Colocalization of Apolipoprotein D proteins and neurofibrillary tangles in the same neuron was rare. Thus, these results suggest that in Alzheimer disease and aging, apolipoprotein D gene expression is increased in stressed cortical neurons before they possibly accumulate neurofibrillary tangles.

Stearoyl-CoA desaturase 1 coding sequences and antisense RNA affect lipid secretion in transfected chicken LMH hepatoma cells.

Hepatic stearoyl CoA desaturase (SCD) activity in chickens from a fat line is higher than that of chickens from a lean line and correlates with plasma triacylglycerol concentrations. Furthermore, in these lines, the hepatic SCD1 mRNA level is positively correlated with the adipose tissue weight. To analyze the contribution of the SCD1 gene in the regulation of adiposity in the early stages of triacylglycerol secretion, SCD1 coding sequence and antisense RNA expression vectors were transfected in LMH cells. After selection, these cells were analyzed with regard to SCD1 expression and lipid secretion. The amounts of secreted triacylglycerols and phospholipids were shown to be higher in LMH cells transfected with the SCD1 gene, but reduced in those transfected with the SCD1 antisense sequences when compared to cells transfected with the vector alone (without SCD1 sequences). These results provide direct evidence that the expression of the SCD1 gene plays a major role in the triacylglycerol and phospholipid secretion process.

Differential messenger RNA distribution of lactate dehydrogenase LDH-1 and LDH-5 isoforms in the rat brain.

The role of lactate in brain energy metabolism has recently received renewed attention. Although blood-borne monocarboxylates such as lactate poorly cross the blood-brain barrier in the adult brain, lactate produced within the brain parenchyma may be a suitable substrate for brain cells. Lactate dehydrogenase is crucial for both the production and utilization of lactate. In this article, we report the regional distribution of the messenger RNAs for lactate dehydrogenase isoforms 1 and 5 in the adult rat brain using in situ hybridization histochemistry with specific [alpha-(35)S]dATP 3' end-labeled oligoprobes. The autoradiographs revealed that the lactate dehydrogenase-1 messenger RNA is highly expressed in a variety of brain structures, including the main olfactory bulb, the piriform cortex, several thalamic and hypothalamic nuclei, the pontine nuclei, the ventral cochlear nucleus, the trigeminal nerve and the solitary tractus nucleus. In addition, the granular and Purkinje cell layers of the cerebellum showed a strong labeling. The neocortex (e.g., cingular, retrosplenial and frontoparietal cortices) often exhibits a marked laminar pattern of distribution of lactate dehydrogenase-1 messenger RNA (layers II/III, IV and VI being most strongly labeled). In contrast, expression of the lactate dehydrogenase-5 messenger RNA generally seemed more diffusely distributed across the different brain regions. Expression was particularly strong in the hippocampal formation (especially in Ammon's horn and dentate gyrus) and in the cerebral cortex, where no laminar pattern of distribution was observed. Overall, these data are consistent with the emerging idea that lactate is an important energy substrate produced and consumed by brain cells.

Triglyceride synthesis and secretion and lipogenesis implicated gene expression in the chicken hepatocarcinoma cell line LMH.

Avian lipogenesis was studied in the chicken hepatocarcinoma LMH cell line. The differentiated and lipogenic status of these cells was evidenced by the presence of the albumin mRNA as well as of some mRNA coding for enzymes involved in lipogenesis (acetyl-CoA carboxylase, fatty acid synthase, delta 9 desaturase) and for apoproteins (apoprotein B and A1). These results were further confirmed by the analysis of triglyceride synthesis and secretion rates in growing cells. A time course analysis showed that triglyceride metabolism was affected by cell density. Hormone responsiveness of triglyceride production was also analyzed. Insulin, triiodothyronine and glucagon to a lesser extent were shown to regulate lipogenesis of LMH cells. The results were compared with those obtained in primary cultures of chicken hepatocytes.

Lipogenic enzyme and apoprotein messenger RNAs in long-term primary culture of chicken hepatocytes.

Hepatocytes isolated from 9-week-old chickens were cultured in a serum-free, hormonally defined medium. Relative amounts of mRNAs coding for lipogenic enzymes (acetyl-CoA carboxylase, fatty acid synthase, delta 9 desaturase, malic enzyme) and apoproteins (apoprotein A1 and apoprotein B) were determined until the 12th day. beta-actin and albumin mRNA, as well as albumin secretion, were also assessed. Cellular metabolic activity appeared to be very low for the first days of culture, but increased after the 7th day. All the mRNAs studied, except for that of malic enzyme, were present from this time throughout the culture lifespan. The biological significance of the observed results and the relevance of this chicken hepatocyte culture system for long-term metabolic and genetic studies are discussed.