Backbone and side-chain 1H, 15N and 13C resonance assignments of the microtubule-binding domain of yeast cytoplasmic dynein in the high and low-affinity states

Cytoplasmic dynein is a motor protein that walks toward the minus end of microtubules (MTs) by utilizing the energy of ATP hydrolysis. The heavy chain of cytoplasmic dynein contains the microtubule-binding domain (MTBD). Switching of MTBD between high and low affinity states for MTs is crucial for processive movement of cytoplasmic dynein. Previous biochemical studies demonstrated that the affinity of MTBD is regulated by the AAA+ family ATPase domain, which is separated by 15 nm long coiled-coil helix. In order to elucidate the structural basis of the affinity switching mechanism of MTBD, we designed two MTBD constructs, termed MTBD-High and MTBD-Low, which are locked in high and low affinity state for MTs, respectively, by introducing a disulfide bond between the coiled-coil helix. Here, we established the backbone and side-chain assignments of MTBD-High and MTBD-Low for further structural analyses.     

Near Infrared Spectroscopy Study of the Frontopolar Hemodynamic Response and Depressive Mood in Children with Major Depressive Disorder: A Pilot Study

AIM

The aim of this study was to evaluate the frontopolar hemodynamic response and depressive mood in children with mild or moderate major depressive disorder during six weeks treatment without medication.

METHODS

The subjects were 10 patients with mild or moderate depression. They were depressive drug-naive children and adolescents. The scores of Depression Self Rating Scale (DSRS), the results of the Verbal Fluency Test (VFT), and the concentrations of oxy-hemoglobin (Oxy-Hb) of frontal pole brain assessed by two-channel near infrared spectroscopy (NIRS) after six weeks of treatment was compared with those of initial treatment.

RESULTS

The score of DSRS was significantly reduced after six weeks of initial treatment (p<0.001, t-test). The word number of VFT was not significantly changed after six weeks of treatment. The oxy-Hb concentration significantly increased after six weeks of treatment (p<0.001, t-test).

CONCLUSIONS

This study demonstrated that the concentration of oxy-Hb of frontopolar cortex in children with mild and moderate depression improved along with their depressive mood. These results suggested that concentration of oxy-Hb using NIRS may be used as the state maker for change in depressive mood of children having depression, similar to that in adults.     

Increase of Hemoglobin Levels by Anti-IL-6 Receptor Antibody (Tocilizumab) in Rheumatoid Arthritis

Objective

To compare the effect of tocilizumab (TCZ) with other biologic therapies in improving anemia of rheumatoid arthritis (RA) patients.

Methods

We compared the change of hemoglobin (Hb) levels in a cohort of 147 consecutive RA patients who were treated with biologics for more than 12 weeks. Twenty eight patients were treated with TCZ, and 119 patients were treated with biologics other than TCZ (87 with TNF inhibitors and 32 with abatacept). The change of Hb levels from baseline to week 12 was compared between the TCZ and the non-TCZ groups. We performed univariate and multivariate analyses with adjustment of potential confounders such as baseline characteristics, concomitant treatment, and the clinical response to treatment.

Results

Hb levels generally increased after biologic therapies both in the TCZ and the non-TCZ groups. The increase of Hb levels was greater in the TCZ group than in the non-TCZ groups (1.1 g/dL in the TCZ group vs 0.3 g/dL in the non-TCZ group, p = 0.009). Univariate analysis revealed that increase of Hb levels was also significantly associated with lower Hb, higher Low Hemoglobin Density, and higher CRP levels at baseline and greater reduction in the clinical disease activity index. TCZ therapy was significantly associated with the increase of Hb levels even after adjustment for these factors by multivariate analysis (p<0.001, effect size 0.08–0.12).

Conclusion

TCZ therapy is an independent factor associated with the increase of Hb level after biologic therapies in RA patients. It will help in selecting appropriate biologics for RA patients with anemia.     

Decrease in the Traumatic Symptoms Observed in Child Survivors within Three Years of the 2011 Japan Earthquake and Tsunami

Background

On March 11, 2011, Japan was struck by a massive earthquake and tsunami. The tsunami caused tremendous damage and traumatized several people, including children. The aim of this study was to assess changes in traumatic symptoms 8, 20, and 30 months of the 2011 tsunami.

Methods

The study comprised three groups. Copies of the Post-Traumatic Stress Symptoms for Children 15 items (PTSSC-15), a self-rating questionnaire on traumatic symptoms, were distributed to 12,524 children (8-month period), 12,193 children (20-month period), and 11,819 children (30-month period). An effective response of children 8 months, 20 months, and 30 month after the disaster was obtained in 11,639 (92.9%), 10,597 (86.9%), and 10,812 children (91.4%), respectively. We calculated the total score, PTSD subscale, and Depression subscale of PTSSC-15. We calculated the total score, PTSD subscale, and Depression subscale of PTSSC-15.

Results

The PTSSC-15 total score and PTSD subscale of children belonging to 1st–9th grade groups who were tested 30 and 20 months after the tsunami significantly decreased compared with those of children tested 8 months after the tsunami. The PTSSC-15 total score and PTSD subscale of children in 1st–9th grade groups tested after 30 months did not decrease significantly compared with those of children tested after 20 months. The PTSSC-15 Depression subscale and PTSD subscale of children in 1st–9th grade groups tested after 30 months significantly decreased compared with those of children tested 8 months after the tsunami. The PTSSC-15 Depression subscale of children in 1st–9th grade groups evaluated after 30 months significantly decreased compared with those of children evaluated after 20 months.

Conclusions

This study demonstrates that the traumatic symptoms of children who survived the massive tsunami improved with time. Nonetheless, the traumatic symptoms, which in some cases did not improve with time.     

Learning/Memory Impairment and Reduced Expression of the HNK-1 Carbohydrate in ��4-Galactosyltransferase-II-deficient Mice

The glycosylation of glycoproteins and glycolipids is important for central nervous system development and function. Although the roles of several carbohydrate epitopes in the central nervous system, including polysialic acid, the human natural killer-1 (HNK-1) carbohydrate, α2,3-sialic acid, and oligomannosides, have been investigated, those of the glycan backbone structures, such as Galβ1-4GlcNAc and Galβ1-3GlcNAc, are not fully examined. Here we report the generation of mice deficient in β4-galactosyltransferase-II (β4GalT-II). This galactosyltransferase transfers Gal from UDP-Gal to a nonreducing terminal GlcNAc to synthesize the Gal β1-4GlcNAc structure, and it is strongly expressed in the central nervous system. In behavioral tests, the β4GalT-II^-/- mice showed normal spontaneous activity in a novel environment, but impaired spatial learning/memory and motor coordination/learning. Immunohistochemistry showed that the amount of HNK-1 carbohydrate was markedly decreased in the brain of β4GalT-II^-/- mice, whereas the expression of polysialic acid was not affected. Furthermore, mice deficient in glucuronyltransferase (GlcAT-P), which is responsible for the biosynthesis of the HNK-1 carbohydrate, also showed impaired spatial learning/memory as described in our previous report, although their motor coordination/learning was normal as shown in this study. Histological examination showed abnormal alignment and reduced number of Purkinje cells in the cerebellum of β4GalT-II^-/- mice. These results suggest that the Galβ1-4GlcNAc structure in the HNK-1 carbohydrate is mainly synthesized by β4GalT-II and that the glycans synthesized by β4GalT-II have essential roles in higher brain functions, including some that are HNK-1-dependent and some that are not.The glycosylation of glycoproteins, proteoglycans, and glycolipids is important for their biological activities, stability, transport, and clearance from circulation, and cell-surface glycans participate in cell-cell and cell-extracellular matrix interactions. In the central nervous system, several specific carbohydrate epitopes, including polysialic acid (PSA),³ the human natural killer-1 (HNK-1) carbohydrate, α2,3-sialic acid, and oligomannosides play indispensable roles in neuronal generation, cell migration, axonal outgrowth, and synaptic plasticity (1). Functional analyses of the glycan backbone structures, like lactosamine core (Galβ1-4GlcNAc), neolactosamine core (Galβ1-3GlcNAc), and polylactosamine (Galβ1-4GlcNAcβ1-3) have been carried out using gene-deficient mice in β4-galactosyltransferase-I (β4GalT-I) (2, 3), β4GalT-V (4), β3-N-acetylglucosaminyl-transferase-II (β3GnT-II) (5), β3GnT-III (Core1-β3GnT) (6), β3GnT-V (7), and Core2GnT (8). However, the roles of these glycan backbone structures in the nervous system have not been examined except the olfactory sensory system (9).β4GalTs synthesize the Galβ1-4GlcNAc structure via the β4-galactosylation of glycoproteins and glycolipids; the β4GalTs transfer galactose (Gal) from UDP-Gal to a nonreducing terminal N-acetylglucosamine (GlcNAc) of N- and O-glycans with a β-1,4-linkage. The β4GalT family has seven members (β4GalT-I to VII), of which at least five have similar Galβ1-4GlcNAc-synthesizing activities (10, 11). Each β4GalT has a tissue-specific expression pattern and substrate specificity with overlapping, suggesting each β4GalT has its own biological role as well as redundant functions. β4GalT-I and β4GalT-II share the highest identity (52% at the amino acid level) among the β4GalTs (12), suggesting these two galactosyltransferases can compensate for each other. β4GalT-I is strongly and ubiquitously expressed in various non-neural tissues, whereas β4GalT-II is strongly expressed in neural tissues (13, 14). Indeed, the β4GalT activity in the brain of β4GalT-I-deficient (β4GalT-I^-/-) mice remains as high as 65% of that of wild-type mice, and the expression levels of PSA and the HNK-1 carbohydrate in the brain of these mice are normal (15). These results suggest β4GalTs other than β4GalT-I, like β4GalT-II, are important in the nervous system.Among the β4GalT family members, only β4GalT-I^-/- mice have been examined extensively; this was done by us and another group. We reported that glycans synthesized by β4GalT-I play various roles in epithelial cell growth and differentiation, inflammatory responses, skin wound healing, and IgA nephropathy development (2, 16-18). Another group reported that glycans synthesized by β4GalT-I are involved in anterior pituitary hormone function and in fertilization (3, 19). However, no other nervous system deficits have been reported in these mice, and the role of the β4-galactosylation of glycoproteins and glycolipids in the nervous system has not been fully examined.In this study, we generated β4GalT-II^-/- mice and examined them for behavioral abnormalities and biochemical and histological changes in the central nervous system. β4GalT-II^-/- mice were impaired in spatial learning/memory and motor coordination/learning. The amount of HNK-1 carbohydrate was markedly decreased in the β4GalT-II^-/- brain, but PSA expression was not affected. These results suggest that the Galβ1-4GlcNAc structure in the HNK-1 carbohydrate is mainly synthesized by β4GalT-II and that glycans synthesized by β4GalT-II have essential roles in higher brain functions, including ones that are HNK-1 carbohydrate-dependent and ones that are independent of HNK-1.     

Role of Vac8 in formation of the vacuolar sequestering membrane during micropexophagy

Vac8 is a yeast vacuolar membrane protein involved in vacuolar membrane dynamics, e.g., vacuole inheritance and vacuolar membrane fusion. This protein is also necessary for a subset of autophagic pathways that deliver specific cellular components to the vacuole. In this study, we show that the micropexohagy and vacuole inheritance required distinct domain structures of Pichia pastoris Vac8 (PpVac8). Whereas vacuole inheritance required the Armadillo repeat (ARM) region that resides in the middle part of the protein, micropexophagy did not. Deletion of both the ARM and C-terminal domains inhibited a characteristic of vacuolar dynamics during micropexophagy, i.e., formation of the vacuolar sequestering membrane (VSM). Subsequent analyses indicated that PpVAC8 disruption abolished recruitment of PpAtg11, another protein required for formation of the VSM, to the vacuolar membrane. These results present a novel molecular function of PpVac8 in micropexophagy.     

Larval arm resorption proceeds concomitantly with programmed cell death during metamorphosis of the sea urchin Hemicentrotus pulcherrimus

Sea urchins are excellent models to elucidate metamorphic phenomena of echinoderms. However, little attention has been paid to the way that their organ resorption is accomplished by programmed cell death (PCD) and related cellular processes. We have used cytohistochemistry and transmission electron microscopy to study arm resorption in competent larvae of metamorphosing sea urchins, Hemicentrotus pulcherrimus, induced to metamorphose by L-glutamine treatment. The results show that: (1) columnar epithelial cells, which are constituents of the ciliary band, undergo PCD in an overlapping fashion with apoptosis and autophagic cell death; (2) squamous epithelial cells, which are distributed between the two arrays of the ciliary band, display a type of PCD distinct from that of columnar epithelial cells, i.e., a cytoplasmic type of non-lysosomal vacuolated cell death; (3) epithelial integrity is preserved even when PCD occurs in constituent cells of the epithelium; (4) secondary mesenchyme cells, probably blastocoelar cells, contribute to the elimination of dying epithelial cells; (5) nerve cells have a delayed initiation of PCD. Taken together, our data indicate that arm resorption in sea urchins proceeds concomitantly with various types of PCD followed by heterophagic elimination, but that epithelial organization is preserved during metamorphosis.This investigation was supported in part by a Keio University special grant-in-aid for innovative collaborative research projects.     

Targeted mutation of serine 697 in the Ret tyrosine kinase causes migration defect of enteric neural crest cells

The RET receptor tyrosine kinase plays a critical role in the development of the enteric nervous system (ENS) and the kidney. Upon glial-cell-line-derived neurotrophic factor (GDNF) stimulation, RET can activate a variety of intracellular signals, including the Ras/mitogen-activated protein kinase, phosphatidylinositol 3-kinase (PI3K)/AKT, and RAC1/JUN NH(2)-terminal kinase (JNK) pathways. We recently demonstrated that the RAC1/JNK pathway is regulated by serine phosphorylation at the juxtamembrane region of RET in a cAMP-dependent manner. To determine the importance of cAMP-dependent modification of the RET signal in vivo, we generated mutant mice in which serine residue 697, a putative protein kinase A (PKA) phosphorylation site, was replaced with alanine (designated S697A mice). Homozygous S697A mutant mice lacked the ENS in the distal colon, resulting from a migration defect of enteric neural crest cells (ENCCs). In vitro organ culture showed an impaired chemoattractant response of the mutant ENCCs to GDNF. JNK activation by GDNF but not ERK, AKT and SRC activation was markedly reduced in neurons derived from the mutant mice. The JNK inhibitor SP600125 and the PKA inhibitor KT5720 suppressed migration of the ENCCs in cultured guts from wild-type mice to comparable degrees. Thus, these findings indicated that cAMP-dependent modification of RET function regulates the JNK signaling responsible for proper migration of the ENCCs in the developing gut.     

Accelerated idioventricular rhythm during ajmaline test: a case report

We present an unusual transient pro-arrhythmic effect of ajmaline in a patient with resuscitated cardiac arrest and a left ventricular apical aneurysm. We discuss the clinical presentation and the possible physio-pathological explanation for this new pro-arrhythmic effect linked to administration of intravenous ajmaline.