Alterations of Regional Cerebral Blood Flow in Tinnitus Patients as Assessed Using Single-Photon Emission Computed Tomography

Tinnitus is the perception of phantom sound without an external auditory stimulus. Using neuroimaging techniques, such as positron emission tomography, electroencephalography, magnetoencephalography, and functional magnetic resonance imaging (fMRI), many studies have demonstrated that abnormal functions of the central nervous system are closely associated with tinnitus. In our previous research, we reported using resting-state fMRI that several brain regions, including the rectus gyrus, cingulate gyrus, thalamus, hippocampus, caudate, inferior temporal gyrus, cerebellar hemisphere, and medial superior frontal gyrus, were associated with tinnitus distress and loudness. To reconfirm these results and probe target regions for repetitive transcranial magnetic stimulation (rTMS), we investigated the regional cerebral blood flow (rCBF) between younger tinnitus patients (<60 years old) and the age-matched controls using single-photon emission computed tomography and easy Z-score imaging system. Compared with that of controls, the rCBF of tinnitus patients was significantly lower in the bilateral medial superior frontal gyri, left middle occipital gyrus and significantly higher in the bilateral cerebellar hemispheres and vermis, bilateral middle temporal gyri, right fusiform gyrus. No clear differences were observed between tinnitus patients with normal and impaired hearing. Regardless of the assessment modality, similar brain regions were identified as characteristic in tinnitus patients. These regions are potentially involved in the pathophysiology of chronic subjective tinnitus.     

The dipeptide prolyl-hydroxyproline promotes cellular homeostasis and lamellipodia-driven motility via active β1-integrin in adult tendon cells

Collagen-derived hydroxyproline (Hyp)-containing peptides have a variety of biological effects on cells. These bioactive collagen peptides are locally generated by the degradation of endogenous collagen in response to injury. However, no comprehensive study has yet explored the functional links between Hyp-containing peptides and cellular behavior. Here, we show that the dipeptide prolyl-4-hydroxyproline (Pro-Hyp) exhibits pronounced effects on mouse tendon cells. Pro-Hyp promotes differentiation/maturation of tendon cells with modulation of lineage-specific factors and induces significant chemotactic activity in vitro. In addition, Pro-Hyp has profound effects on cell proliferation, with significantly upregulated extracellular signal–regulated kinase phosphorylation and extracellular matrix production and increased type I collagen network organization. Using proteomics, we have predicted molecular transport, cellular assembly and organization, and cellular movement as potential linked-network pathways that could be altered in response to Pro-Hyp. Mechanistically, cells treated with Pro-Hyp demonstrate increased directional persistence and significantly increased directed motility and migration velocity. They are accompanied by elongated lamellipodial protrusions with increased levels of active β1-integrin–containing focal contacts, as well as reorganization of thicker peripheral F-actin fibrils. Pro-Hyp–mediated chemotactic activity is significantly reduced (p < 0.001) in cells treated with the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or the α5β1-integrin antagonist ATN-161. Furthermore, ATN-161 significantly inhibits uptake of Pro-Hyp into adult tenocytes. Thus, our findings document the molecular basis of the functional benefits of the Pro-Hyp dipeptide in cellular behavior. These dynamic properties of collagen-derived Pro-Hyp dipeptide could lead the way to its application in translational medicine.     

Structural analysis of N-glycans of the planarian Dugesia japonica

To investigate the relationship between phylogeny and glycan structures, we analyzed the structure of planarian N-glycans. The planarian Dugesia japonica, a member of the flatworm family, is a lower metazoan. N-glycans were prepared from whole worms by hydrazinolysis, followed by tagging with the fluorophore 2-aminopyridine at their reducing end. The labeled N-glycans were purified, and separated by three HPLC steps. By comparison with standard pyridylaminated N-glycans, it was shown that the N-glycans of planarian include high mannose-type and pauci-mannose-type glycans. However, many of the major N-glycans from planarians have novel structures, as their elution positions did not match those of the standard glycans. The results of mass spectrometry and sugar component analyses indicated that these glycans include methyl mannoses, and that the most probable linkage was 3-O-methylation. Furthermore, the methyl residues on the most abundant glycan may be attached to the non-reducing-end mannose, as the glycans were resistant to α-mannosidase digestion. These results indicate that methylated high-mannose-type glycans are the most abundant structure in planarians.     

Analysis of tryptophan‐rich region in Clostridium septicum alpha‐toxin involved with binding to glycosylphosphatidylinositol‐anchored proteins

Clostridium septicum alpha‐toxin has a unique tryptophan‐rich region (³⁰²NGYSEWDWKWV³¹²) that consists of 11 amino acid residues near the C‐terminus. Using mutant toxins, the contribution of individual amino acids in the tryptophan‐rich region to cytotoxicity and binding to glycosylphosphatidylinositol (GPI)‐anchored proteins was examined. For retention of maximum cytotoxic activity, W307 and W311 are essential residues and residue 309 has to be hydrophobic and possess an aromatic side chain, such as tryptophan or phenylalanine. When residue 308, which lies between tryptophans (W307 and W309) is changed from an acidic to a basic amino acid, the cytotoxic activity of the mutant is reduced to less than that of the wild type. It was shown by a toxin overlay assay that the cytotoxic activity of each mutant toxin correlates closely with affinity to GPI‐anchored proteins. These findings indicate that the WDW_W sequence in the tryptophan‐rich region plays an important role in the cytotoxic mechanism of alpha‐toxin, especially in the binding to GPI‐anchored proteins as cell receptors.     

Chewing number is related to incremental increases in body weight from 20 years of age in Japanese middle‐aged adults

doi: 10.1111/j.1741‐2358.2012.00666.x Chewing number is related to incremental increases in body weight from 20 years of age in Japanese middle‐aged adults Background: Eating habits are associated with both current obesity and incremental increases in body weight from young adulthood, but no study has focused on chewing number during meals among community residents. Objective: This study focused on the relationship between chewing number and incremental increases in body weight from 20 years of age. Methods: A total of 93 persons aged 35–61 years participated. The subjects were asked to set the device and record their chewing number during each meal on a particular day. They were also asked whether their body weight had increased by 10 kg or more since they were 20 years old. Results: The body weight of 28 subjects (30%) had increased more than 10 kg since the age of 20 years. Total chewing number showed a relationship with such body weight increases. The odds ratio of weight increments of more than 10 kg for the lowest tertile group was 4.6 [95% confidence interval (CI), 1.3–16.2] relative to the highest tertile group (Model 1). The odds ratio of weight increments for the lowest tertile group increased to 6.3 (95% CI, 1.6–25.4) in Model 2 and to 9.1 (95% CI, 1.7–49.8) in Model 3. Conclusion: Although this study was limited because it did not consider all risk factors, categorical chewing number was related independently to body weight increments of more than 10 kg from 20 years of age.     

Elucidation of biosynthetic pathway of a plant hormone abscisic acid in phytopathogenic fungi

ABSTRACT

Abscisic acid (ABA) is one of the plant hormones that regulates physiological functions in various organisms, including plants, sponges, and humans. The biosynthetic machinery in plants is firmly established, while that in fungi is still unclear. Here, we elucidated the functions of the four biosynthetic genes, bcABA1-bcABA4, found in Botrytis cinerea by performing biotransformation experiments and in vitro enzymatic reactions with putative biosynthetic intermediates. The first-committed step is the cyclization of farnesyl diphosphate to give α-ionylideneethane catalyzed by a novel sesquiterpene synthase, BcABA3, which exhibits low amino acid sequence identities with sesquiterpene synthases. Subsequently, two cytochrome P450s, BcABA1 and BcABA2, mediate oxidative modifications of the cyclized product to afford 1?,4?-trans-dihydroxy-α-ionylideneacetic acid, which undergoes alcohol oxidation to furnish ABA. Our results demonstrated that production of ABA does not depend on the nucleotide sequence of bcABA genes. The present study set the stage to investigate the role of ABA in infections.     

Streptomycin-phosphorylating Enzyme Produced by Streptomyces griseus

Streptomycin-phosphorylating enzyme was reported previously to be produced in mycelium of Streptomyces griseus HUT 6037 at late stage of growth. In the present investigation, this enzyme was purified 200 times as high in specific activity as cell-free extract by means of salting out, chromatography on DEAE-Sephadex A-25 and gel filtration with Sephadex G-100. This enzyme was most stable at pH 8.0 and required 10^?2mMg²⁺ in the reaction mixture for the highest activity. It lost the activity by heat treatment at 40°C for 15 min in absence of the substrate.

Mutant cultures were prepared on productivity of or tolerance to streptomycin, and their capacity to produce streptomycin-phosphorylating enzyme was examined. The cultures which had low to no capacity to produce streptomycin produced a small amount to none of the enzyme, suggesting that production of the streptomycin-phosphorylating enzyme had some correlation with streptomycin productivity of the culture. But no definite correlation was observed between productivity of the enzyme and the capacity to tolerate streptomycin.     

Translocation domain of botulinum neurotoxin A subtype 2 potently induces entry into neuronal cells

Botulinum neurotoxin (BoNT) is the causative agent of botulism in humans and animals. Only BoNT serotype A subtype 1 (BoNT/A1) is used clinically because of its high potency and long duration of action. BoNT/A1 and BoNT/A subtype 2 (BoNT/A2) have a high degree of amino acid sequence similarity in the light chain (LC) (96%), whereas their N-and C-terminal heavy chain (H_N and H_C) differ by 13%. The LC acts as a zinc-dependent endopeptidase, H_N as the translocation domain, and H_C as the receptor-binding domain. BoNT/A2 and BoNT/A1 had similar potency in the mouse bioassay, but BoNT/A2 entered faster and more efficiently into neuronal cells. To identify the domains responsible for these characteristics, H_N of BoNT/A1 and BoNT/A2 was exchanged to construct chimeric BoNT/A121 and BoNT/A212. After expression in Escherichia coli, chimeric and wild-type BoNT/As were purified as single-chain proteins and activated by conversion to disulfide-linked dichains. The toxicities of recombinant wild-type and chimeric BoNT/As were similar, but dropped to 60% compared with the values of native BoNT/As. The relative orders of SNAP-25 cleavage activity in neuronal cells and toxicity differed. BoNT/A121 and recombinant BoNT/A2 have similar SNAP-25 cleavage activity. BoNT/A2 H_N is possibly responsible for the higher potency of BoNT/A2 than BoNT/A1.