Permeability of the blood-brain barrier to a novel satiety molecule nesfatin-1

Nesfatin-1 has recently been identified as a hypothalamic and brain stem peptide that regulates feeding behavior. Here, we determined the ability of nesfatin-1 to cross the blood–brain barrier (BBB) of mice. We used multiple-regression analysis to determine that radioactively labeled nesfatin-1 injected intravenously entered the brain. The entry rate (K_i) of ¹³¹I-nesfatin-1 from blood-to-brain was 0.20 ± 0.02 μl/g min. This modest rate of entry was not inhibited by the administration of nonradioactive nesfatin-1, suggesting that BBB transport of nesfatin-1 into the brain is by a nonsaturable mechanism. High performance liquid chromatography (HPLC) and acid precipitation showed that most of the injected radiolabeled nesfatin-1 reached the brain as intact peptide, and capillary depletion with vascular washout revealed that 67% of ¹³¹I-nesfatin-1 crossed the BBB to reach the brain parenchyma. Efflux of labeled nesfatin-1 from brain back into blood was by way of bulk flow. These findings demonstrate that nesfatin-1 crosses the BBB in both the blood-to-brain and brain-to-blood directions by nonsaturable mechanisms.     

A protective association between catalase and isocitrate lyase in peroxisomes

Glyoxysomes are specialized peroxisomes in germinating seeds, which catalyze many reactions that convert fatty acids into carbohydrates thus generating H(2)O(2). They are characterized by the presence of catalase (CAT, E.C. 1.11.1.6) in their matrix which protects cells from oxidative stress. Here, we investigated the possibility that a protein can be protected from oxidative damage by its association with CAT. We purified peroxisomal CAT from germinating castor beans by ion exchange, gel filtration, and hydroxylapatite chromatography. Gel filtration of the matrix proteins, cross-linking, and co-immunoprecipitation studies indicate that CAT associates with a glyoxysomal matrix protein, isocitrate lyase (ICL, E.C. 4.1.3.1). In addition, we found that H(2)O(2) inactivates ICL and degrades its product, glyoxylate, when CAT is inactive. ICL and its product appear to be sensitive to oxidative damage; thus, association of CAT with ICL would afford protection from H(2)O(2).     

The role of peripheral chemoreceptor activity on the respiratory responses to hypoxia and hypercapnia in anaesthetised rabbits with induced hypothyroidism

The purpose of this study was to investigate the role of peripheral chemoreceptor activity on the hypoxic and hypercapnic ventilatory drives in rabbits with induced hypothyroidism. Experiments were carried out in control and hypothyroid rabbits. Hypothyroidism was induced by an administration of an iodide-blocker, methimazole in food (75 mg/100 g food) for ten weeks. At the end of the tenth week, triiodothyronine (T3) and thyroxine (T4) levels significantly decreased (P<0.001) while thyroid stimulating hormone (TSH) increased (P<0.001). Tidal volume (VT), respiratory frequency (f/min), ventilation minute volume (VE) and systemic arterial blood pressure (BP) were recorded during the breathing of the normoxic, hypoxic (8% O2-92% N2) and hypercapnic (6% CO2-Air) gas mixtures, in the anaesthetised rabbits of both groups. At the end of each experimental phase, PaO2, PaCO2, and pHa were measured. The same experimental procedure was repeated after peripheral chemoreceptor denervation in both groups. VT significantly decreased in some of the rabbits with hypothyroidism during the breathing of the hypoxic gas mixture (nonresponsive subgroup) (P<0.05). After chemodenervation, a decrease in VT was observed in this nonresponsive subgroup during normoxia (P<0.05). The percent decrease in VT in nonresponsive subgroup of hypothyroid rabbits after chemodenervation was lower than that of the chemodenervated control animals (P<0.01). When these rabbits with hypothyroidism were allowed to breath the hypercapnic gas mixtures, increases in VT and VE were not significant. In conclusion, although there is a decrease in peripheral chemoreceptor activity in hypothyroidism, it does not seem to be the only cause of decrease in ventilatory drive during hypoxia and hypercapnia.     

Fine structure DNA mapping studies of the chromosomal region harboring the genetic defect in neurofibromatosis type 1

To better map the location of the von Recklinghausen neurofibromatosis (NF1) gene, we have characterized a somatic cell hybrid designated 7AE-11. This microcell-mediated, chromosome-transfer construct harbors a centromeric segment and a neo-marked segment from the distal long arm of human chromosome 17. We have identified 269 cosmid clones with human sequences from a 7AE-11 library and, using a panel of somatic cell hybrids with a total of six chromosome 17q breakpoints, have mapped 240 of these clones on chromosome 17q. The panel included a hybrid (NF13) carrying a der(22) chromosome that was isolated from an NF1 patient with a balanced translocation, t(17;22) (q11.2;q11.2). Fifty-three of the cosmids map into a region spanning the NF13 breakpoint, as defined by the two closest flanking breakpoints (17q11.2 and 17q11.2-q12). RFLP clones from a subset of these cosmids have been mapped by linkage analysis in normal reference families, to localize the NF1 gene more precisely and to enhance the potential for genetic diagnosis of this disorder. The cosmids in the NF1 region will be an important resource for testing DNA blots of large-fragment restriction-enzyme digests from NF1 patient cell lines, to detect rearrangements in patients' DNA and to identify the 17;22 NF1 translocation breakpoint.     

Distribution ofFrankia in soils from forest and afforestation sites in northern Sweden

Summary The presence in soil ofFrankia, capable of forming nitrogen-fixing root nodules onAlnus incana (L.) Moench, was investigated. Intact soil cores from forested as well as disturbed sites were sampled and both alder-rich and alder-free sites were included in the study. Surface-sterilized alder seeds were sown in the soil cores which were kept in sterile culture tubes in a growth chamber. Root nodules with nitrogenase activity developed in soil cores from all sites studied. Thus, infective and effectiveFrankia was present in all of the soils sampled, even from sites free from actinorhizal plants and irrespective of pH and nitrogen content of the soils.     

A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom

Research in yeast and animals has resulted in a well-supported consensus model for eukaryotic cell cycle control. The fit of this model to early diverging eukaryotes, such as the plant kingdom, remains unclear. Using the green alga Chlamydomonas reinhardtii, we developed an efficient pipeline, incorporating robotics, semiautomated image analysis, and deep sequencing, to molecularly identify >50 genes, mostly conserved in higher plants, specifically required for cell division but not cell growth. Mutated genes include the cyclin-dependent kinases CDKA (resembling yeast and animal Cdk1) and the plant-specific CDKB. The Chlamydomonas cell cycle consists of a long G1 during which cells can grow >10-fold, followed by multiple rapid cycles of DNA replication and segregation. CDKA and CDKB execute nonoverlapping functions: CDKA promotes transition between G1 and entry into the division cycle, while CDKB is essential specifically for spindle formation and nuclear division, but not for DNA replication, once CDKA-dependent initiation has occurred. The anaphase-promoting complex is required for similar steps in the Chlamydomonas cell cycle as in Opisthokonts; however, the spindle assembly checkpoint, which targets the APC in Opisthokonts, appears severely attenuated in Chlamydomonas, based on analysis of mutants affecting microtubule function. This approach allows unbiased integration of the consensus cell cycle control model with innovations specific to the plant lineage.     

Single-molecule analysis of the microtubule cross-linking protein MAP65-1 reveals a molecular mechanism for contact-angle-dependent microtubule bundling

Bundling of microtubules (MTs) is critical for the formation of complex MT arrays. In land plants, the interphase cortical MTs form bundles specifically following shallow-angle encounters between them. To investigate how cells select particular MT contact angles for bundling, we used an in vitro reconstitution approach consisting of dynamic MTs and the MT-cross-linking protein MAP65-1. We found that MAP65-1 binds to MTs as monomers and inherently targets antiparallel MTs for bundling. Dwell-time analysis showed that the affinity of MAP65-1 for antiparallel overlapping MTs is about three times higher than its affinity for single MTs and parallel overlapping MTs. We also found that purified MAP65-1 exclusively selects shallow-angle MT encounters for bundling, indicating that this activity is an intrinsic property of MAP65-1. Reconstitution experiments with mutant MAP65-1 proteins with different numbers of spectrin repeats within the N-terminal rod domain showed that the length of the rod domain is a major determinant of the range of MT bundling angles. The length of the rod domain also determined the distance between MTs within a bundle. Together, our data show that the rod domain of MAP65-1 acts both as a spacer and as a structural element that specifies the MT encounter angles that are conducive for bundling.