LIMK1 regulates Golgi dynamics, traffic of Golgi-derived vesicles, and process extension in primary cultured neurons

In this study, we examined the subcellular distribution and functions of LIMK1 in developing neurons. Confocal microscopy, subcellular fractionation, and expression of several epitope-tagged LIMK1 constructs revealed that LIMK1 is enriched in the Golgi apparatus and growth cones, with the LIM domain required for Golgi localization and the PDZ domain for its presence at neuritic tips. Overexpression of wild-type LIMK1 suppresses the formation of trans-Golgi derived tubules, and prevents cytochalasin D-induced Golgi fragmentation, whereas that of a kinase-defective mutant has the opposite effect. Transfection of wild-type LIMK1 accelerates axon formation and enhances the accumulation of Par3/Par6, insulin-like growth factor (IGF)1 receptors, and neural cell adhesion molecule (NCAM) at growth cones, while inhibiting the Golgi export of synaptophysin-containing vesicles. These effects were dependent on the Golgi localization of LIMK1, paralleled by an increase in cofilin phosphorylation and phalloidin staining in the region of the Golgi apparatus, and prevented by coexpression of constitutive active cofilin. The long-term overexpression of LIMK1 produces growth cone collapse and axon retraction, an effect that is dependent on its growth cone localization. Together, our results suggest an important role for LIMK1 in axon formation that is related with its ability to regulate Golgi dynamics, membrane traffic, and actin cytoskeletal organization.     

Age-dependent decreases of phosphorus and magnesium in human achilles’ tendons

To elucidate changes of human tendons with aging, the authors studied age-related changes of elements in human Achilles’ tendons by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of seven men and seven women, ranging in age from 61 to 97 yr. It was found that the content of calcium increased progressively with aging in the Achilles’ tendons, whereas the contents of phosphorus and magnesium decreased gradually with aging. The previous investigations demonstrated that the content of calcium and phosphorus increased progressively with aging in most, but not all, human tissues, except for the bones. In ligaments, such as the anterior cruciate ligament and the ligament of the head of the femur, which are histologically similar to the Achilles’ tendon, it was previously found that both the contents of calcium and phosphorus increased with aging in the ligaments. It should be noted that the content of phosphorus in the Achilles’ tendons decreased during the aging process. In addition, it was found that there was a very high direct correlation between phosphorus and magnesium contents in the tendons, but not between calcium and phosphorus contents.     

Influence of light and darkness on the ultrastructure of the pineal organ in the blind cave fish,Astyanax mexicanus

Ultrastructural changes of the pineal organ were investigated in the blind cave fish, Astyanax mexicanus, kept under continous artificial light (5000 lux), in continuous darkness, and under natural light conditions. The pineal end-vesicle of the fish kept under natural photoperiod consisted of photoreceptor cells and supporting cells mixed with a few ganglion cells. The photoreceptor cells possessed well-developed outer segments with regularly arranged lamellar membranes. The supporting cells contained a number of lipid droplets and large globular cisternae filled with fine granules. In the fish kept under continuous light or in darkness, the pineal end-vesicle displayed a dilated lumen, and the outer segments of the receptors showed signs of degeneration. Furthermore, alterations of cell organelles were observed in the photoreceptor and supporting cells.     

Neural Networks for Mindfulness and Emotion Suppression

Mindfulness, an attentive non-judgmental focus on “here and now” experiences, has been incorporated into various cognitive behavioral therapy approaches and beneficial effects have been demonstrated. Recently, mindfulness has also been identified as a potentially effective emotion regulation strategy. On the other hand, emotion suppression, which refers to trying to avoid or escape from experiencing and being aware of one’s own emotions, has been identified as a potentially maladaptive strategy. Previous studies suggest that both strategies can decrease affective responses to emotional stimuli. They would, however, be expected to provide regulation through different top-down modulation systems. The present study was aimed at elucidating the different neural systems underlying emotion regulation via mindfulness and emotion suppression approaches. Twenty-one healthy participants used the two types of strategy in response to emotional visual stimuli while functional magnetic resonance imaging was conducted. Both strategies attenuated amygdala responses to emotional triggers, but the pathways to regulation differed across the two. A mindful approach appears to regulate amygdala functioning via functional connectivity from the medial prefrontal cortex, while suppression uses connectivity with other regions, including the dorsolateral prefrontal cortex. Thus, the two types of emotion regulation recruit different top-down modulation processes localized at prefrontal areas. These different pathways are discussed.     

The first representative of glycosylated three-fingered toxins. Cytotoxin from the Naja kaouthia cobra venom.

There are different glycosylated proteins in snake venoms, but no glycosylated representatives of a large family of three-fingered toxins have previously been detected. A new glycoprotein was isolated from the venom of the Thai cobra Naja kaouthia. MALDI MS of the glycoprotein contained an array of peaks in the range from approximately 8900 to approximately 9400 Da indicating its microheterogeneity. Carbohydrate analysis showed the presence of mannose, galactose, N-acetylglucosamine, fucose and neuraminic acid. The N-terminal sequence of the glycoprotein was identical to that of cytotoxin 3 (CX3) from N. kaouthia, and CD spectra of the glycoprotein and CX3 were almost the same. Cleavage of a glycan moiety by N-glycosidase F gave a protein of molecular mass practically coinciding with that of CX3. MALDI MS of the tryptic digest of reduced glycoprotein S-pyridylethylated at cysteine residues, contained peaks corresponding to all tryptic fragments of CX3, with the exception of fragment 24-30. The peak corresponding to this peptide appeared in the mass-spectrum of similarly treated deglycosylated glycoprotein. These data show that the potential N-glycosylation site at Asn29 in CX3 is utilized for glycan attachment and that the glycoprotein is glycosylated CX3. In vivo toxicity of the glycoprotein to the cricket Gryllus assimilis was twofold lower than that of CX3. The cytotoxic activity of the glycoprotein towards HL60 cells was about two orders of magnitude lower than that of CX3, but could be made equal to the CX3 cytotoxicity by deglycosylation. Thus for the first time we have isolated a glycosylated three-fingered snake venom toxin wherein glycosylation appears to modulate its biological activity.     

A new regulation of IL-6 production in adult cardiomyocytes by β-adrenergic and IL-1β receptors and induction of cellular hypertrophy by IL-6 trans-signalling

The sympathetic nervous system and pro-inflammatory cytokines play key roles in numerous cardiovascular disorders. Chronic β-adrenergic receptor (β-AR) stimulation in myocardium induces expression of pro-inflammatory cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6), which contribute to cardiac hypertrophy and failure. To evaluate the relationship between β-AR stimulation and pro-inflammatory cytokines, we studied the effects of the β-AR agonist isoprenaline (ISO) on IL-1-induced IL-6 production in adult rat ventricular myocytes (ARVMs). We report that ISO and IL-1 synergistically enhanced IL-6 gene expression and secretion. The synergistic effect of ISO was mimicked by cAMP elevating agents and involved the G_s protein/cAMP/PKA signalling pathway, but not the exchange factor EPAC. To evaluate the contribution of IL-6 to cellular hypertrophy, we examined the signalling pathways stimulated by the membrane-bound IL-6 receptor (IL-6R), and the IL-6 soluble receptor (sIL-6R) involved in the mechanism named IL-6 trans-signalling. The IL-6/sIL-6R complex promoted a rapid and persistent phosphorylation of STAT3^Tyr705 in ARVMs. Moreover, IL-6 trans-signalling increased protein synthesis, c-fos gene expression and B-type natriuretic peptide secretion, three markers of cardiac hypertrophy. IL-6 trans-signalling also increased cell size. In contrast, IL-6 alone had no significant effect on either cell size or STAT3 phosphorylation although it induced phosphorylation of ERK1/2, AKT and S6K, demonstrating the presence of a functional IL-6R in ARVMs. Taken together, these results demonstrate that β-AR stimulation synergises with IL-1 for IL-6 secretion in adult ventricular myocytes and indicate that IL-6 induces cardiac hypertrophy only via IL-6 trans-signalling. The IL-6 soluble receptor may thus serve as a switch for IL-6 to activate STAT3 phosphorylation and hypertrophy.     

Discovery of 2-substituted benzoxazole carboxamides as 5-HT3 receptor antagonists

A new class of 2-substituted benzoxazole carboxamides are presented as potent functional 5-HT(3) receptor antagonists. The chemical series possesses nanomolar in vitro activity against human 5-HT(3)A receptors. A chemistry optimization program was conducted and identified 2-aminobenzoxazoles as orally active 5-HT(3) receptor antagonists with good metabolic stability. These novel analogues possess drug-like characteristics and have potential utility for the treatment of diseases attributable to improper 5-HT(3) receptor function, especially diarrhea predominant irritable bowel syndrome (IBS-D).