The defective protein level of myosin light chain phosphatase (MLCP) in the isolated saphenous vein, as a vascular conduit in coronary artery bypass grafting (CABG), harvested from patients with diabetes mellitus (DM)

We examined the contractile reactivity to 5-hydroxytryptamine (5-HT) in isolated human saphenous vein (SV), as a vascular conduit in coronary artery bypass grafting (CABG), harvested from patients with diabetes mellitus (DM) and non-DM (NDM). Vascular rings of endothelium-denuded SV were used for functional and biochemical experiments. The vasoconstrictions caused by 5-HT were significantly greater (hyperreactivity) in the DM group than in the NDM group. RhoA/ROCK pathway is activated by various G-protein-coupled receptor agonists and consequently induces phosphorylation of myosin phosphatase target subunit 1 (MYPT1), a subunit of myosin light chain phosphatase (MLCP), which inhibits MLCP activity. In the resting state of the vessels, total tissue protein levels of 5-HT_2A receptor, 5-HT_1B receptor, RhoA, ROCK1, and ROCK2 did not differ between NDM and DM groups. However, the total protein level of MYPT1 was significantly lower in the DM group than in the NDM group. Furthermore, the ratio of P(Thr⁶⁹⁶)-MYPT1 to total MYPT1 was significantly higher in the DM group than in the NDM group. These results suggest that the hyperreactivity to 5-HT in the SV smooth muscle of patients with DM is due to not only enhanced phosphorylation of MLCP but also defective protein level of MLCP. Thus, we reveal for the first time that the defective protein level of MLCP in the DM group can partially explain the poor patency of SV graft harvested from patients with DM.     

Fission yeast Swi5-Sfr1 protein complex, an activator of Rad51 recombinase, forms an extremely elongated dogleg-shaped structure

In eukaryotes, DNA strand exchange is the central reaction of homologous recombination, which is promoted by Rad51 recombinases forming a right-handed nucleoprotein filament on single-stranded DNA, also known as a presynaptic filament. Accessory proteins known as recombination mediators are required for the formation of the active presynaptic filament. One such mediator in the fission yeast Schizosaccharomyces pombe is the Swi5-Sfr1 complex, which has been identified as an activator of Rad51 that assists in presynaptic filament formation and stimulates its strand exchange reaction. Here, we determined the 1:1 binding stoichiometry between the two subunits of the Swi5-Sfr1 complex using analytical ultracentrifugation and electrospray ionization mass spectrometry. Small-angle x-ray scattering experiments revealed that the Swi5-Sfr1 complex displays an extremely elongated dogleg-shaped structure in solution, which is consistent with its exceptionally high frictional ratio (f/f(0)) of 2.0 ± 0.2 obtained by analytical ultracentrifugation. Furthermore, we determined a rough topology of the complex by comparing the small-angle x-ray scattering-based structures of the Swi5-Sfr1 complex and four Swi5-Sfr1-Fab complexes, in which the Fab fragments of monoclonal antibodies were specifically bound to experimentally determined sites of Sfr1. We propose a model for how the Swi5-Sfr1 complex binds to the Rad51 filament, in which the Swi5-Sfr1 complex fits into the groove of the Rad51 filament, leading to an active and stable presynaptic filament.     

Genetic Analysis of Putative Triploid Miscanthus Hybrids and Tetraploid M. sacchariflorus Collected from Sympatric Populations of Kushima, Japan

Miscanthus ?×giganteus, a triploid hybrid between tetraploid M. sacchariflorus and diploid M. sinensis, has considerable potential as a bioenergy crop. Currently only one genotype is widely cultivated, increasing its vulnerability to diseases during production. Finding new hybrids is important to broaden genetic resources of M. ×giganteus. Three putative triploid hybrids were discovered in a sympatric population of tetraploid M. sacchariflorus and diploid M. sinensis in Kushima, Japan. The hybrid nature of the triploids was determined by morphological analysis and sequencing the ribosomal DNA internal transcribed spacer (ITS) region. The triploids had awns on their florets, which is a common characteristic of diploid M. sinensis, and sheath hairs, which is typical of tetraploid M. sacchariflorus. All triploids showed heterozygosity in their ribosomal DNA ITS sequences. Based on these results, it is confirmed that the triploids are hybrids and novel genotypes of M. ×giganteus. Natural crossing between tetraploid M. sacchariflorus × diploid M. sinensis may also lead to the production of tetraploid hybrids. ITS analysis of tetraploid plants showed that one maternal parent of the triploid hybrids, K-Ogi-1, had heterozygous ITS, which was different than the other analyzed tetraploid, M. sacchariflorus. Thus, K-Ogi-1 was likely of hybrid origin. These tetraploid hybrids can also be utilized as parents in M. ×giganteus breeding. Since all hybrids identified in this study had tetraploid M. sacchariflorus as maternal parents, collecting and analyzing seeds from tetraploid M. sacchariflorus in sympatric areas could be an effective strategy to identify natural Miscanthus hybrids that can be used as bioenergy crops.     

Severity and Patterns of Blood-Nerve Barrier Breakdown in Patients with Chronic Inflammatory Demyelinating Polyradiculoneuropathy: Correlations with Clinical Subtypes

Objective

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is currently classified into clinical subtypes, including typical and atypical forms (multifocal acquired demyelinating sensory and motor neuropathy (MADSAM) and distal acquired demyelinating symmetric neuropathy (DADS)). The aim of this study was to elucidate the patterns and severity of breakdown of the blood-nerve barrier (BNB) in each CIDP subtype.

Methods

We evaluated the effects of sera obtained from patients with typical CIDP, MADSAM and DADS and control subjects on the expression levels of tight junction proteins and transendothelial electrical resistance (TEER) value in human peripheral nerve microvascular endothelial cells (PnMECs).

Results

The sera obtained from the patients with the three clinical phenotypes of CIDP decreased the amount of claudin-5 protein levels and TEER values in the PnMECs. In addition, the sera obtained from typical CIDP patients more prominently reduced claudin-5 protein levels and TEER values in the PnMECs than did that obtained from the MADSAM and DADS patients. Furthermore, the severity of BNB disruption after exposure to the sera was associated with higher Hughes grade, lower MRC score, more pronounced slowing of motor nerve conduction in the median nerve and higher frequency of abnormal temporal dispersion.

Conclusions

Sera derived from typical CIDP patients destroy the BNB more severely than those from MADSAM or DADS patients. The extent of BNB disruption in the setting of CIDP is associated with clinical disability and demyelination in the nerve trunk. These observations may explain the phenotypical differences between CIDP subtypes.     

Cardiac-Specific Inhibition of Kinase Activity in Calcium/Calmodulin-Dependent Protein Kinase Kinase-β Leads to Accelerated Left Ventricular Remodeling and Heart Failure after Transverse Aortic Constriction in Mice

Background

The mechanism of cardiac energy production against sustained pressure overload remains to be elucidated.

Methods and Results

We generated cardiac-specific kinase-dead (kd) calcium/calmodulin-dependent protein kinase kinase-β (CaMKKβ) transgenic (α-MHC CaMKKβ^kd TG) mice using α-myosin heavy chain (α-MHC) promoter. Although CaMKKβ activity was significantly reduced, these mice had normal cardiac function and morphology at baseline. Here, we show that transverse aortic binding (TAC) in α-MHC CaMKKβ^kd TG mice led to accelerated death and left ventricular (LV) dilatation and dysfunction, which was accompanied by significant clinical signs of heart failure. CaMKKβ downstream signaling molecules, including adenosine monophosphate-activated protein kinase (AMPK), were also suppressed in α-MHC CaMKKβ^kd TG mice compared with wild-type (WT) mice. The expression levels of peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, which is a downstream target of both of CaMKKβ and calcium/calmodulin kinases, were also significantly reduced in α-MHC CaMKKβ^kd TG mice compared with WT mice after TAC. In accordance with these findings, mitochondrial morphogenesis was damaged and creatine phosphate/β-ATP ratios assessed by magnetic resonance spectroscopy were suppressed in α-MHC CaMKKβ^kd TG mice compared with WT mice after TAC.

Conclusions

These data indicate that CaMKKβ exerts protective effects on cardiac adaptive energy pooling against pressure-overload possibly through phosphorylation of AMPK and by upregulation of PGC-1α. Thus, CaMKKβ may be a therapeutic target for the treatment of heart failure.     

Neuroanatomical technique for studying long axonal projections in the central nervous system: combined axonal staining and pre-labeling in parasagittal gerbil brain slices

A method is described for studying the morphological features of extensive axonal projections within the central nervous system of the gerbil, Meriones anguiculatus. Potentially long descending axonal projections between the auditory thalamus and lower brainstem were used as a model. The inferior colliculus (IC) in the tectum was injected in vivo with a fluorescent retrograde tracer, Fluoro-Gold, to label cells in the medial geniculate body (MGB) that had descending projections to the IC, and cells in the superior olivary complex (SOC) that had ascending projections to the IC. Another fluorescent retrograde tracer, fast blue, was injected into the cochlea to label olivocochlear (OC) cells in the SOC. Inferomedially curved parasagittal slices containing ipsilateral auditory cell groups from the thalamus to the brainstem were cut and descending axons of the pre-labeled MGB cells were traced anterogradely with Biocytin. After visualizing histologically the injected Biocytin, discretely labeled IC-projecting axons of the MGB cells were traced including their collaterals that extended further into the SOC. In the SOC, these axons terminated on pre-labeled cells including OC cells. The combination of anterograde and retrograde tracing in the slice preparations described here demonstrated extensive descending axonal projections from the thalamus to their targets in the lower brainstem that had known ascending/descending projections within the auditory system.     

Reaction System for Violaxanthin De-Epoxidase with PSII Membranes

PSII membranes were used as a substrate for violaxanthin de-epoxidase(VDE) that had been solubilized from spinach thylakoids by sonication.Inclusion of Tween 20 in the assay mixture was essential, althoughthe detergent apparently inhibited the activity in the conventionalassay with purified violaxanthin and lipid as substrate. Themaximum enhancing effect of the detergent was observed nearits critical micellar concentration. It is likely that the monomerof the detergent helped VDE react with the substrate in themembranes. Dependence of the activity on the substrate concentrationsuggested that VDE functions at least at two sites in the membranes,probably on both their lumenal and stromal surfaces. The abilityof the enzyme to function on the stromal surface in in vitroassays was demonstrated by using intact thylakoids as the substrate.Under such conditions where the endogenous VDE was functioningin the lumen, the exogenously added VDE converted an-theraxanthinto zeaxanthin in the absence of Tween 20. This result suggeststhat, in the reaction with PSII membranes, the detergent wasrequired for VDE to react with violaxanthin but not with antheraxanthin.Otherwise, the detergent was necessary for the reaction on thelumenal surface. (Received September 5, 1997; Accepted October 19, 1997)     

Molecular cloning and characterization of the family of feline leucine-rich glioma-inactivated (LGI) genes,and mutational analysis in familial spontaneous epileptic cats

Background

Leucine-rich glioma-inactivated (LGI) proteins play a critical role in synaptic transmission. Dysfunction of these genes and encoded proteins is associated with neurological disorders such as genetic epilepsy or autoimmune limbic encephalitis in animals and human. Familial spontaneous epileptic cats (FSECs) are the only feline strain and animal model of familial temporal lobe epilepsy. The seizure semiology of FSECs comprises recurrent limbic seizures with or without evolution into generalized epileptic seizures, while cats with antibodies against voltage-gated potassium channel complexed/LGI1 show limbic encephalitis and recurrent limbic seizures. However, it remains unclear whether the genetics underlying FSECs are associated with LGI family genes. In the present study, we cloned and characterized the feline LGI1–4 genes and examined their association with FSECs. Conventional PCR techniques were performed for cloning and mutational analysis. Characterization was predicted using bioinformatics software.

Results

The cDNAs of feline LGI1–4 contained 1674-bp, 1650-bp, 1647-bp, and 1617-bp open reading frames, respectively, and encoded proteins comprising 557, 549, 548, and 538 amino acid residues, respectively. The feline LGI1–4 putative protein sequences showed high homology with Homo sapiens, Canis familiaris, Bos taurus, Sus scrofa, and Equus caballus (92%–100%). Mutational analysis in 8 FSECs and 8 controls for LGI family genes revealed 3 non-synonymous and 14 synonymous single nucleotide polymorphisms in the coding region. Only one non-synonymous single nucleotide polymorphism in LGI4 was found in 3 out of 8 FSECs. Using three separate computational tools, this mutation was not predicted to be disease causing. No co-segregation of the disease was found with any variant.

Conclusions

We cloned the cDNAs of the four feline LGI genes, analyzed the amino acid sequences, and revealed that epilepsy in FSEC is not a monogenic disorder associated with LGI genes.