Complementary roles for Nkx6 and Nkx2 class proteins in the establishment of motoneuron identity in the hindbrain

The genetic program that underlies the generation of visceral motoneurons in the developing hindbrain remains poorly defined. We have examined the role of Nkx6 and Nkx2 class homeodomain proteins in this process, and provide evidence that these proteins mediate complementary roles in the specification of visceral motoneuron fate. The expression of Nkx2.2 in hindbrain progenitor cells is sufficient to mediate the activation of Phox2b, a homeodomain protein required for the generation of hindbrain visceral motoneurons. The redundant activities of Nkx6.1 and Nkx6.2, in turn, are dispensable for visceral motoneuron generation but are necessary to prevent these cells from adopting a parallel program of interneuron differentiation. The expression of Nkx6.1 and Nkx6.2 is further maintained in differentiating visceral motoneurons, and consistent with this the migration and axonal projection properties of visceral motoneurons are impaired in mice lacking Nkx6.1 and/or Nkx6.2 function. Our analysis provides insight also into the role of Nkx6 proteins in the generation of somatic motoneurons. Studies in the spinal cord have shown that Nkx6.1 and Nkx6.2 are required for the generation of somatic motoneurons, and that the loss of motoneurons at this level correlates with the extinguished expression of the motoneuron determinant Olig2. Unexpectedly, we find that the initial expression of Olig2 is left intact in the caudal hindbrain of Nkx6.1/Nkx6.2 compound mutants, and despite this, all somatic motoneurons are missing. These data argue against models in which Nkx6 proteins and Olig2 operate in a linear pathway, and instead indicate a parallel requirement for these proteins in the progression of somatic motoneuron differentiation. Thus, both visceral and somatic motoneuron differentiation appear to rely on the combined activity of cell intrinsic determinants, rather than on a single key determinant of neuronal cell fate.     

Specificity of human anti-NOR antibodies,a distinct species of "natural" anti-alpha-galactosyl antibodies

Natural anti-NOR antibodies are common in human sera and agglutinate human erythrocytes of a rare NOR phenotype. The NOR phenotype-related antigens are unique neutral glycosphingolipids recognized by these antibodies and Griffonia simplicifolia IB4 isolectin (GSL-IB4). The oligosaccharide chains of NOR glycolipids are terminated by Galalpha1-4GalNAcbeta1-3Galalpha units. To characterize the specificity of anti-NOR antibodies and compare it with specificities of GSL-IB4 and known anti-Galalpha1,3Gal antibodies, alpha-galactosylated saccharides and saccharide-polyacrylamide conjugates were used. New synthetic oligosaccharides, corresponding to the terminal di- and trisaccharide sequence of NOR glycolipids and the conjugate of the NOR-tri with HSA were included. These compounds were tested by microtiter plate ELISA and hemagglutination inhibition. Anti-NOR antibodies reacted most strongly with Galalpha1-4GalNAcbeta1-3Gal (NOR-tri), and over 100 times less strongly with Galalpha1-4GalNAc (NOR-di). The antibodies reacted also with Galalpha1-4Gal and Galalpha1-4Galbeta1-4GlcNAc, similarly as with NOR-di but not with other tested compounds. In turn, anti-Galalpha1,3Gal antibodies reacted most strongly with Galalpha1-3Gal and were very weakly inhibited by the NOR-related oligosaccharides (weaker than by galactose), and NOR-tri was less active than NOR-di. GSL-IB4 reacted with all tested alpha-galactosylated saccharides and conjugates, including the similarly active NOR-tri and NOR-di. These results showed that anti-NOR represent a new species of anti-alpha-galactosyl antibodies with high affinity for the Galalpha1-4GalNAcbeta1-3Gal sequence present in rare NOR erythrocytes.     

Frankia KB5 Possesses a Hydrogenase Immunologically Related to Membrane-Bound [NiFe]-Hydrogenases

The immunological relationship of the hydrogenase in Frankia KB5 to hydrogenases in other microorganisms was investigated using antisera raised against holo-[NiFe]-hydrogenases isolated from Alcaligenes latus, Azotobacter vinelandii, Ralstonia eutropha, and the small and large hydrogenase subunits from Bradyrhizobium japonicum. The antisera raised against the A. latus, R. eutropha, and B. japonicum (large subunit) polypeptides were found to recognize two polypeptides, corresponding to the unprocessed and processed forms of the hydrogenase subunit in Frankia KB5. None of the antisera, including the antibodies produced against the small hydrogenase subunit isolated from B. japonicum, recognized any polypeptide related to the small hydrogenase subunit in Frankia KB5. An immunogold localization study of the intracellular distribution of hydrogenase in Frankia KB5, with the cryo-section technique, showed that labeling in the membrane of both hyphae and vesicles was positively correlated with hydrogenase activity. Received: 6 November 2000 / Accepted: 18 December 2000     

Deactivation of TSH receptor signaling in filter-cultured pig thyroid epithelial cells

Thyrotropin [thyroid-stimulating hormone (TSH)] receptor on-off signaling was studied in polarized monolayers of pig thyrocytes cultured on permeable support. Transepithelial resistance (R) and potential difference (PD) were used as parameters to monitor the effect of altered TSH concentrations on vectorial electrolyte transport. TSH induced rapid but long-lasting changes in R (decrease) and PD (increase) that were cAMP-dependent and related to enhanced transcellular conductance of sodium and chloride. Withdrawal of TSH from cultures prestimulated with TSH (0.1 mU/ml) for 48 h resulted in restitution of R to control level within 30 min. Such deactivation was markedly accelerated by mild trypsinization, which degraded receptor-bound ligand without affecting TSH receptor responsiveness or ion transporting capacity. Small alterations in the TSH concentration (0.01-0.1 mU/ml) were followed almost instantaneously by adjustments of R. In contrast, the reversal of R after acute TSH stimulation (30 min) and subsequent TSH washout was delayed for several hours independently of cell surface trypsinization. The observations indicate that, during continuous exposure to physiological concentrations, TSH exerts a close minute-to-minute surveillance of thyroid function and the rate-limiting step of deactivation is the dissociation of ligand from the TSH receptor at the cell surface. TSH-deprived cells briefly exposed to TSH are refractory to rapid deactivation, probably because of altered metabolism downstream of TSH receptor signal transduction.     

Simultaneous detection of spin-coupled and decoupled QA– EPR-signals in Photosystem II complexes isolated with isoelectric focusing

The Photosystem II multisubunit protein complex can be extracted from thylakoid membranes with non-ionic detergents and subjected to various spectroscopical and biochemical investigations. This paper shows that after extraction with dodecyl--D-maltoside, several Photosystem II complexes could be resolved by isoelectric focusing. Structurally, the various Photosystem II complexes differed from each other in polypeptide composition, especially with regard to the chlorophyll a/b-binding proteins, which gave rise to differing isoelectric points. Functionally, the various Photosystem II complexes differed from each other on the acceptor side, as judged by acceptor side-dependent electron transfer and electron paramagnetic resonance (EPR). The Q_A ^- Fe2+-signal (g = 1.84), arising from Q_A ^- spin-coupled to the acceptor-side iron, and a radical signal arising from decoupled Q_A ^- (g = 2.0045) could be detected simultaneously in some of the Photosystem II complexes, and the amount of each of the two signals were inversely related. The results are discussed in relation to previously known heterogeneities in Photosystem II.     

The Association between Carbohydrate-Rich Foods and Risk of Cardiovascular Disease Is Not Modified by Genetic Susceptibility to Dyslipidemia as Determined by 80 Validated Variants

Background

It is still unclear whether carbohydrate consumption is associated with cardiovascular disease (CVD) risk. Genetic susceptibility might modify the associations between dietary intakes and disease risk.

Objectives

The aim was to examine the association between the consumption of carbohydrate-rich foods (vegetables, fruits and berries, juice, potatoes, whole grains, refined grains, cookies and cakes, sugar and sweets, and sugar-sweetened beverages) and the risk of incident ischemic CVD (iCVD; coronary events and ischemic stroke), and whether these associations differ depending on genetic susceptibility to dyslipidemia.

Methods

Among 26,445 individuals (44–74 years; 62% females) from the Malmö Diet and Cancer Study cohort, 2,921 experienced an iCVD event during a mean follow-up time of 14 years. At baseline, dietary data were collected using a modified diet history method, and clinical risk factors were measured in 4,535 subjects. We combined 80 validated genetic variants associated with triglycerides and HDL-C or LDL-C, into genetic risk scores and examined the interactions between dietary intakes and genetic risk scores on the incidence of iCVD.

Results

Subjects in the highest intake quintile for whole grains had a 13% (95% CI: 3–23%; p-trend: 0.002) lower risk for iCVD compared to the lowest quintile. A higher consumption of foods rich in added sugar (sugar and sweets, and sugar-sweetened beverages) had a significant cross-sectional association with higher triglyceride concentrations and lower HDL-C concentrations. A stronger positive association between a high consumption of sugar and sweets on iCVD risk was observed among those with low genetic risk score for triglycerides (p-interaction=0.05).

Conclusion

In this prospective cohort study that examined food sources of carbohydrates, individuals with a high consumption of whole grains had a decreased risk of iCVD. No convincing evidence of an interaction between genetic susceptibility for dyslipidemia, measured as genetic risk scores of dyslipidemia-associated variants, and the consumption of carbohydrate-rich foods on iCVD risk was observed.     

A Novel 1,4-Dihydropyridine Derivative Improves Spatial Learning and Memory and Modifies Brain Protein Expression in Wild Type and Transgenic APPSweDI Mice

Ca²⁺ blockers, particularly those capable of crossing the blood-brain barrier (BBB), have been suggested as a possible treatment or disease modifying agents for neurodegenerative disorders, e.g., Alzheimer’s disease. The present study investigated the effects of a novel 4-(N-dodecyl) pyridinium group-containing 1,4-dihydropyridine derivative (AP-12) on cognition and synaptic protein expression in the brain. Treatment of AP-12 was investigated in wild type C57BL/6J mice and transgenic Alzheimer’s disease model mice (Tg APP_SweDI) using behavioral tests and immunohistochemistry, as well as mass spectrometry to assess the blood-brain barrier (BBB) penetration. The data demonstrated the ability of AP-12 to cross the BBB, improve spatial learning and memory in both mice strains, induce anxiolytic action in transgenic mice, and increase expression of hippocampal and cortical proteins (GAD67, Homer-1) related to synaptic plasticity. The compound AP-12 can be seen as a prototype molecule for use in the design of novel drugs useful to halt progression of clinical symptoms (more specifically, anxiety and decline in memory) of neurodegenerative diseases, particularly Alzheimer’s disease.