Spontaneous Recovery of the Injured Higher Olfactory Center in the Terrestrial Slug Limax

Background

Of all organs and tissues in adult mammals, the brain shows the most limited regeneration and recovery after injury. This is one reason why treating neurological damage such as ischemic injury after stroke presents such a challenge. Here we report a novel mode of regeneration which the slug''s cognitive center, the procerebrum, shows after surgical lesioning in the adult. It is well known that the land slug Limax possesses the capacity to demonstrate conditioned food aversion. This learning ability critically depends on the procerebrum, which is the higher olfactory center in the brain of the terrestrial mollusk.

Principal Findings

In the present study, after a 1-month recovery period post-surgical lesioning of the procerebrum we investigated whether the brain of the slug shows recovery from damage. We found that learning ability, local field potential oscillation, and the number of cells in the procerebrum (PC) all recovered spontaneously within 1 month of bilateral lesioning of the PC. Moreover, neurogenesis was enhanced in the lesioned PC. However, memory acquired before the surgery could not be retrieved 1 month after surgery although the procerebrum had recovered from injury by this time, consistent with the notion that the procerebrum is the storage site of odor-aversion memory, or deeply involved in the memory recall process.

Significance

Our findings are the first to demonstrate that a brain region responsible for the associative memory of an adult organism can spontaneously reconstitute itself, and can recover its function following injury.     

Myeloid Differentiation Factor 88 (MyD88)-Deficiency Increases Risk of Diabetes in Mice

Background

Multiple lines of evidence suggest innate immune response pathways to be involved in the development of obesity-associated diabetes although the molecular mechanism underling the disease is unknown. Recent observations suggest that saturated fatty acids can act as a ligand for toll-like receptor (TLR) 4, which is thought to mediate obesity-associated insulin resistance. Myeloid differentiation factor 88 (MyD88) is an adapter protein for TLR/IL-1 receptor signaling, which is involved in the activation of inflammatory pathways. To evaluate molecular mechanisms linking obesity-associated diabetes down-stream of TLR4, we investigated physiological role of MyD88 in high-fat diet (HFD)-induced obesity.

Methodology/Principal Findings

In the present study, we found MyD88-deficient mice fed a HFD had increased circulating levels of insulin, leptin and cholesterol, as well as liver dysfunction (increased induction of ALT levels, increased activation of JNK and cleavage of PARP), which were linked to the onset of severe diabetes. On the other hand, TNF-α would not be involved in HFD-induced diabetes in MyD88-deficient mice, because TNF-α level was attenuated in MyD88-deficient mice fed with HFD.

Conclusions/Significance

The present finding of an unexpected role for MyD88 in preventing diabetes may provide a potential novel target/strategy for treating metabolic syndrome.     

Characterization and sequence of tomato 2S seed albumin: a storage protein with sequence similarities to the fruit lectin

We found a 2S storage albumin from the seed of tomato ( Lycopersicon esculentum L. cv. Cherry) that cross-reacted with antiserum to the fruit lectin, and named it Lec2SA. According to its size and basicity, Lec2SA was classified into four isoforms. These isoforms have an M(r) of approximately 12,000, and are composed of a small subunit (M(r) 4,000) and a large subunit (M(r) 8,000) linked by disulfide bonds. The complete amino acid sequence of Lec2SA was determined. The small subunit was composed of 32 amino acids, whereas the large subunit contained 70 amino acids with a pyroglutamine as the N-terminal residue. The sequence of Lec2SA was similar to that of 2S albumins from different plants, such as Brazil nut and castor beans. Furthermore, a sequence similarity was found between the large subunit of Lec2SA and the peptide sequence from tomato lectin. Although these similarities were found, Lec2SA did not show hemagglutinating activity or sugar-chain-binding activity, indicating that Lec2SA lacks the carbohydrate-binding domain. These results suggest that tomato lectin is a chimeric lectin sharing the seed storage protein-like domain that is incorporated into the gene encoding tomato lectin through gene fusion.     

Glucose activates H(+)-ATPase in kidney epithelial cells

The vacuolar H⁺-ATPase (V-ATPase) acidifies compartments of the vacuolar system of eukaryotic cells. In renal epithelial cells, it resides on the plasma membrane and is essential for bicarbonate transport and acid-base homeostasis. The factors that regulate the H⁺-ATPase remain largely unknown. The present study examines the effect of glucose on H⁺-ATPase activity in the pig kidney epithelial cell line LLC-PK₁. Cellular pH was measured by performing ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. Intracellular acidification was induced with NH₃/NH₄⁺ prepulse, and rates of intracellular pH (pH_i) recovery (after in situ calibration) were determined by the slopes of linear regression lines during the first 3 min of recovery. The solutions contained 1 µM ethylisopropylamiloride and were K⁺ free to eliminate Na⁺/H⁺ exchange and H⁺-K⁺-ATPase activity. After NH₃/NH₄⁺-induced acidification, LLC-PK₁ cells had a significant pH_i recovery rate that was inhibited entirely by 100 nM of the V-ATPase inhibitor concanamycin A. Acute removal of glucose from medium markedly reduced V-ATPase-dependent pH_i recovery activity. Readdition of glucose induced concentration-dependent reactivation of V-ATPase pH_i recovery activity within 2 min. Glucose replacement produced no significant change in cell ATP or ADP content. H⁺-ATPase activity was completely inhibited by the glycolytic inhibitor 2-deoxy-D-glucose (20 mM) but only partially inhibited by the mitochondrial electron transport inhibitor antimycin A (20 µM). The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (500 nM) abolished glucose activation of V-ATPase, and activity was restored after wortmannin removal. Glucose activates V-ATPase activity in kidney epithelial cells through the glycolytic pathway by a signaling pathway that requires PI3K activity. These findings represent an entirely new physiological effect of glucose, linking it to cellular proton secretion and vacuolar acidification. proton secretion; glycolysis; intracellular pH; concanamycin A     

Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate

Staining of esterase-active bacteria with carboxyfluorescein diacetate (CFDA) has been used to evaluate the viability of various types of cell. However, the outer membrane of Gram-negative bacteria prevents CFDA from permeating into the cell. Although EDTA can increase the permeability of the outer membrane allowing CFDA to enter the cells, it was experimentally confirmed that there is still considerable difficulty in visualizing viable cells due to passive diffusion of carboxyfluorescein (CF), a hydrolyzed product of CFDA, out of the cells. We found that glutaraldehyde enhances the discriminative recognition of esterase-active Gram-negative bacteria under microscopic observation by improving the efficacy of staining. We believe the successful staining in the presence of glutaraldehyde is due to two separate effects: an increase in the permeability of CFDA into the cell and prevention of leakage of CF out of the cell.     

Growth inhibitory alkaloids from mesquite (Prosopis juliflora (Sw.) DC.) leaves

Plant growth inhibitory alkaloids were isolated from the extract of mesquite [Prosopis juliflora (Sw.) DC.] leaves. Their chemical structures were established by ESI-MS, 1H and 13C NMR spectra analysis. The I50 value (concentration required for 50% inhibition of control) for root growth of cress (Lepidium sativum L.) seedlings was 400 microM for 3'-oxo-juliprosopine, 500 microM for secojuliprosopinal, and 100 microM for a (1:1) mixture of 3-oxo-juliprosine and 3'-oxo-juliprosine, respectively. On the other hand, the minimum concentration exhibiting inhibitory effect on shoot growth of cress seedlings was 10 microM for 3'-oxo-juliprosopine, 100 microM for secojuliprosopinal, and 1 microM for a (1:1) mixture of 3-oxo-juliprosine and 3'-oxo-juliprosine, respectively. Among these compounds, a (1:1) mixture of 3-oxo-juliprosine and 3'-oxo-juliprosine exhibited the strongest inhibitory effect on the growth of cress seedlings.     

Cytochrome P450-catalyzed brassinosteroid pathway activation through synthesis of castasterone and brassinolide in Phaseolus vulgaris

The last reaction in the biosynthesis of brassinolide has been examined enzymatically. A microsomal enzyme preparation from cultured cells of Phaseolus vulgaris catalyzed a conversion from castasterone to brassinolide, indicating that castasterone 6-oxidase (brassinolide synthase) is membrane associated. This enzyme preparation also catalyzed the conversions of 6-deoxocastasterone and typhasterol to castasterone which have been reported to be catalyzed by cytochrome P450s, CYP85A1 of tomato and CYP92A6 of pea, respectively. The activities of these enzymes require molecular oxygen as well as NADPH as a cofactor. The enzyme activities were strongly inhibited by carbon monoxide, an inhibitor of cytochrome P450, and this inhibition was recovered by blue light irradiation in the presence of oxygen. Commercial cytochrome P450 inhibitors including cytochrome c, SKF 525A, 1-aminobenzotriazole and ketoconazole also inhibited the enzyme activities. The present work presents unanimous enzymological evidence that cytochrome P450s are responsible for the synthesis of brassinolide from castasterone as well as of castasterone from typhasterol and 6-deoxocastasterone, which have been deemed activation steps of BRs.     

Novel biosynthetic pathway of castasterone from cholesterol in tomato

Endogenous brassinosteroids (BRs) in tomato (Lycopersicon esculentum) seedlings are known to be composed of C27- and C28-BRs. The biosynthetic pathways of C27-BRs were examined using a cell-free enzyme solution prepared from tomato seedlings that yielded the biosynthetic sequences cholesterol --> cholestanol and 6-deoxo-28-norteasterone <--> 6-deoxo-28-nor-3-dehydroteasterone <--> 6-deoxo-28-nortyphasterol --> 6-deoxo-28-norcastasterone --> 28-norcastasterone (28-norCS). Arabidopsis CYP85A1 that was heterologously expressed in yeast mediated the conversion of 6-deoxo-28-norCS to 28-norCS. The same reaction was catalyzed by an enzyme solution from wild-type tomato but not by an extract derived from a tomato dwarf mutant with a defect in CYP85. Furthermore, exogenously applied 28-norCS restored the abnormal growth of the dwarf mutant. These findings indicate that the C-6 oxidation of 6-deoxo-28-norCS to 28-norCS in tomato seedlings is catalyzed by CYP85, just as in the conversion of 6-deoxoCS to CS. Additionally, the cell-free solution also catalyzed the C-24 methylation of 28-norCS to CS in the presence of NADPH and S-adenosylmethionine (SAM), a reaction that was clearly retarded in the absence of NADPH and SAM. Thus it seems that C27-BRs, in addition to C28-BRs, are important in the production of more active C28-BRs and CS, where a SAM-dependent sterol methyltransferase appears to biosynthetically connect C27-BRs to C28-BRs. Moreover, the tomato cell-free solution converted CS to 26-norCS and [2H6]CS to [2H3]28-norCS, suggesting that C-28 demethylation is an artifact due to an isotope effect. Although previous feeding experiments employing [2H6]CS suggested that 28-norCS was synthesized from CS in certain plant species, this is not supported in planta. Altogether, this study demonstrated for the first time, to our knowledge, that 28-norCS is not synthesized from CS but from cholesterol. In addition, CS and [2H6]CS were not converted into BL and [2H6]BL, respectively, confirming an earlier finding that the active BR in tomato seedlings is not BL but CS. In conclusion, the biosynthesis of 28-norBRs appears to play a physiologically important role in maintaining homeostatic levels of CS in tomato seedlings.     

Hippocampal cytochrome P450s synthesize brain neurosteroids which are paracrine neuromodulators of synaptic signal transduction

Hippocampal pyramidal neurons and granule neurons of adult male rats are equipped with a complete machinery for the synthesis of pregnenolone, dehydroepiandrosterone, 17beta-estradiol and testosterone as well as their sulfate esters. These brain neurosteroids are synthesized by cytochrome P450s (P450scc, P45017alpha and P450arom) from endogenous cholesterol. Synthesis is acutely dependent on the Ca(2+) influx attendant upon neuron-neuron communication via N-methyl-D-aspartate (NMDA) receptors. Pregnenolone sulfate, estradiol and corticosterone rapidly modulate neuronal signal transduction and the induction of long-term potentiation via NMDA receptors and putative membrane steroid receptors. Brain neurosteroids are therefore promising neuromodulators that may either activate or inactivate neuron-neuron communication, thereby mediating learning and memory in the hippocampus.     

A rice brassinosteroid-deficient mutant, ebisu dwarf (d2), is caused by a loss of function of a new member of cytochrome P450

We characterized a rice dwarf mutant, ebisu dwarf (d2). It showed the pleiotropic abnormal phenotype similar to that of the rice brassinosteroid (BR)-insensitive mutant, d61. The dwarf phenotype of d2 was rescued by exogenous brassinolide treatment. The accumulation profile of BR intermediates in the d2 mutants confirmed that these plants are deficient in late BR biosynthesis. We cloned the D2 gene by map-based cloning. The D2 gene encoded a novel cytochrome P450 classified in CYP90D that is highly similar to the reported BR synthesis enzymes. Introduction of the wild D2 gene into d2-1 rescued the abnormal phenotype of the mutants. In feeding experiments, 3-dehydro-6-deoxoteasterone, 3-dehydroteasterone, and brassinolide effectively caused the lamina joints of the d2 plants to bend, whereas more upstream compounds did not cause bending. Based on these results, we conclude that D2/CYP90D2 catalyzes the steps from 6-deoxoteasterone to 3-dehydro-6-deoxoteasterone and from teasterone to 3-dehydroteasterone in the late BR biosynthesis pathway.