Racemization of the substrate and product by serine palmitoyltransferase from Sphingobacterium multivorum yields two enantiomers of the product from d-serine.

Serine palmitoyltransferase (SPT) catalyzes the pyridoxal-5'-phosphate (PLP)-dependent decarboxylative condensation of l-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (KDS). Although SPT was shown to synthesize corresponding products from amino acids other than l-serine, it is still arguable whether SPT catalyzes the reaction with d-serine, which is a question of biological importance. Using high substrate and enzyme concentrations, KDS was detected after the incubation of SPT from Sphingobacterium multivorum with d-serine and palmitoyl-CoA. Furthermore, the KDS comprised equal amounts of 2S and 2R isomers. 1H-NMR study showed a slow hydrogen-deuterium exchange at Cα of serine mediated by SPT. We further confirmed that SPT catalyzed the racemization of serine. The rate of the KDS formation from d-serine was comparable to those for the α-hydrogen exchange and the racemization reaction. The structure of the d-serine-soaked crystal (1.65 Å resolution) showed a distinct electron density of the PLP-l-serine aldimine, interpreted as the racemized product trapped in the active site. The structure of the α-methyl-d-serine-soaked crystal (1.70 Å resolution) showed the PLP-α-methyl-d-serine aldimine, mimicking the d-serine-SPT complex prior to racemization. Based on these enzymological and structural analyses, the synthesis of KDS from d-serine was explained as the result of the slow racemization to l-serine, followed by the reaction with palmitoyl-CoA, and SPT would not catalyze the direct condensation between d-serine and palmitoyl-CoA. It was also shown that the S. multivorum SPT catalyzed the racemization of the product KDS, which would explain the presence of (2R)-KDS in the reaction products.

Structural insights into the substrate recognition of serine palmitoyltransferase from Sphingobacterium multivorum.

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis, which catalyzes the pyridoxal-5'-phosphate-dependent decarboxylative condensation reaction of l-serine (l-Ser) and palmitoyl-CoA (PalCoA) to form 3-ketodihydrosphingosine called long chain base (LCB). SPT is also able to metabolize l-alanine (l-Ala) and glycine (Gly), albeit with much lower efficiency. Human SPT is a membrane-bound large protein complex containing SPTLC1/SPTLC2 heterodimer as the core subunits, and it is known that mutations of the SPTLC1/SPTLC2 genes increase the formation of deoxy-type of LCBs derived from l-Ala and Gly to cause some neurodegenerative diseases. In order to study the substrate recognition of SPT, we examined the reactivity of Sphingobacterium multivorum SPT on various amino acids in the presence of PalCoA. The S. multivorum SPT could convert not only l-Ala and Gly but also l-homoserine, in addition to l-Ser, into the corresponding LCBs. Furthermore, we obtained high-quality crystals of the ligand-free form and the binary complexes with a series of amino acids, including a nonproductive amino acid, l-threonine, and determined the structures at 1.40 to 1.55 Å resolutions. The S. multivorum SPT accommodated various amino acid substrates through subtle rearrangements of the active-site amino acid residues and water molecules. It was also suggested that non-active-site residues mutated in the human SPT genes might indirectly influence the substrate specificity by affecting the hydrogen-bonding networks involving the bound substrate, water molecules, and amino acid residues in the active site of this enzyme. Collectively, our results highlight SPT structural features affecting substrate specificity for this stage of sphingolipid biosynthesis.

Crystal structure of Sphingobacterium multivorum serine palmitoyltransferase complexed with tris(hydroxymethyl)aminomethane.

Serine palmitoyltransferase (SPT) catalyses the first reaction in sphingolipid biosynthesis: the decarboxylative condensation of L-serine (L-Ser) and palmitoyl-CoA to form 3-ketodihydrosphingosine. SPT from Sphingobacterium multivorum has been isolated and its crystal structure in complex with L-Ser has been determined at 2.3 Šresolution (PDB entry 3a2b). However, the quality of the crystal was not good enough to judge the conformation of the cofactor molecule and the orientations of the side chains of the amino-acid residues in the enzyme active site. The crystal quality was improved by revision of the purification procedure and by optimization of both the crystallization procedure and the post-crystallization treatment conditions. Here, the crystal structure of SPT complexed with tris(hydroxymethyl)aminomethane (Tris), a buffer component, was determined at 1.65 Šresolution. The protein crystallized at 20°C and diffraction data were collected from the crystals to a resolution of 1.65 Å. The crystal belonged to the tetragonal space group P41212, with unit-cell parameters a = b = 61.32, c = 208.57 Å. Analysis of the crystal structure revealed C4-C5-C5A-O4P (77°) and C5-C5A-O4P-P (-143°) torsion angles in the phosphate-group moiety of the cofactor pyridoxal 5'-phosphate (PLP) that are more reasonable than those observed in the previously reported crystal structure (14° and 151°, respectively). Furthermore, the clear electron density showing a Schiff-base linkage between PLP and the bulky artificial ligand Tris indicated exceptional flexibility of the active-site cavity of this enzyme. These findings open up the possibility for further study of the detailed mechanisms of substrate recognition and catalysis by this enzyme.

Ossified Metaplastic Spinal Meningioma Without Psammomatous Calcification: A Case Report.

Meningiomas constitute approximately 25% of primary spinal cord tumors, and 1% to 5% are calcified. Ossification is a rare event and the etiology of ossification in meningiomas is not well known. We present the case of a 29-year-old female with a rare case of ossified thoracic spinal metaplastic meningioma. The tumor was successfully resected, and pathology confirmed ossified metaplastic meningioma. On histopathological examination, only mature bone tissue and tumor cells were present in the region containing no psammoma bodies, suggesting that the tumor cells had transitioned to mature osteocytes.

Change in the pennation angle of the supraspinatus muscle after rotator cuff tear repair.

BACKGROUND: The pennation angle is an important architectural and functional feature of pennate muscles. The purpose of this study was to investigate the change in the pennation angle of the supraspinatus muscle after rotator cuff tear repair. MATERIALS AND METHODS: The study included 68 patients who underwent arthroscopic rotator cuff repair and magnetic resonance imaging. The size of the tear was measured under arthroscopic visualization. The pennation angle of the supraspinatus both preoperatively and postoperatively and the integrity of the repaired cuff were determined by magnetic resonance imaging. RESULTS: The preoperative pennation angle was significantly greater with enlargement of the tear size (P < .0001, analysis of variance). The retear rate was 29% in patients with medium tears and 59% in patients with large or massive tears. No retear was noted in patients with partial and small tears. The retear rate was 90.9% when the preoperative pennation angle was 20° or greater and was 12.3% when this angle was 19° or less, and the risk ratio for retear was 7.4 when this angle was 20° or greater. For repair-type tears, comparison between the preoperative and postoperative pennation angles showed a significant decrease in the mean value from 11.8° ± 3.7° to 9.9° ± 3.0° in the medium tear group (P = .007, paired t test) but no significant difference in the large or massive tear group (from 15.1° ± 7.0° to 13.3° ± 5.8°) (P = .33). For retear-type tears, no significance was found between groups. CONCLUSION: The preoperative pennation angle is directly correlated with the tear configuration and could be one of the prognostic factors for postoperative cuff integrity. To restore the pennation angle, primary repair is more appropriate in smaller rotator cuff tears than in medium-sized tears.

Significance of Stabilometry for Assessing Postoperative Body Sway in Patients with Cervical Myelopathy.

STUDY DESIGN: Prospective study. PURPOSE: To examine the changes in body sway using stabilometry in patients who underwent cervical laminoplasty for cervical myelopathy. OVERVIEW OF LITERATURE: Although the patients of cervical myelopathy complain body sway there are few report to examine body sway objectively. METHODS: Patients who received treatment for cervical myelopathy between October 2010 and February 2013 were included. Twenty-one patients underwent cervical laminoplasty (myelopathy group). Body sway was assessed using stabilometry, wherein patients stood on a stabilometer with their eyes closed for 30 seconds. The Romberg ratio, outer peripheral area (OPA) with eyes closed (cm2), and total locus length per unit area (L/A) with eyes closed (/cm) were examined. Examinations were performed preoperatively (at baseline) and at 8 weeks postoperatively. Examination results of patients in the myelopathy group were compared with those of 17 healthy individuals (control group). Clinical symptoms were evaluated using the Japanese Orthopaedic Association scale score (JOA score) and the timed up and go (TUG) test. RESULTS: In the myelopathy and control groups, the mean baseline Romberg ratio, OPA, and L/A were 2.3±1.2, 8.9±5.5 cm2, and 14.2±5.3/cm and 1.4±1.0, 4.3±2.8 cm2, and 23.7±10.1/cm, respectively. Eight weeks after laminoplasty, only L/A showed significant improvement from baseline in the myelopathy group (23.2±10.1 to 16.8±7.9; p=0.03). The Romberg ratio and OPA showed improvement in the myelopathy group, but the changes were not statistically significant. JOA scores and TUG test results in this group significantly improved from baseline to 8 weeks after laminoplasty (12.7 to 13.4 and 10.8 to 8.0 seconds, respectively; both p<0.05). CONCLUSIONS: L/A is a useful parameter for measuring body sway to assess the recovery of body sway after laminoplasty.

rgs-CaM Detects and Counteracts Viral RNA Silencing Suppressors in Plant Immune Priming.

Primary infection of a plant with a pathogen that causes high accumulation of salicylic acid in the plant typically via a hypersensitive response confers enhanced resistance against secondary infection with a broad spectrum of pathogens, including viruses. This phenomenon is called systemic acquired resistance (SAR), which is a plant priming for adaption to repeated biotic stress. However, the molecular mechanisms of SAR-mediated enhanced inhibition, especially of virus infection, remain unclear. Here, we show that SAR against cucumber mosaic virus (CMV) in tobacco plants (Nicotiana tabacum) involves a calmodulin-like protein, rgs-CaM. We previously reported the antiviral function of rgs-CaM, which binds to and directs degradation of viral RNA silencing suppressors (RSSs), including CMV 2b, via autophagy. We found that rgs-CaM-mediated immunity is ineffective against CMV infection in normally growing tobacco plants but is activated as a result of SAR induction via salicylic acid signaling. We then analyzed the effect of overexpression of rgs-CaM on salicylic acid signaling. Overexpressed and ectopically expressed rgs-CaM induced defense reactions, including cell death, generation of reactive oxygen species, and salicylic acid signaling. Further analysis using a combination of the salicylic acid analogue benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and the Ca2+ ionophore A23187 revealed that rgs-CaM functions as an immune receptor that induces salicylic acid signaling by simultaneously perceiving both viral RSS and Ca2+ influx as infection cues, implying its autoactivation. Thus, secondary infection of SAR-induced tobacco plants with CMV seems to be effectively inhibited through 2b recognition and degradation by rgs-CaM, leading to reinforcement of antiviral RNA silencing and other salicylic acid-mediated antiviral responses.IMPORTANCE Even without an acquired immune system like that in vertebrates, plants show enhanced whole-plant resistance against secondary infection with pathogens; this so-called systemic acquired resistance (SAR) has been known for more than half a century and continues to be extensively studied. SAR-induced plants strongly and rapidly express a number of antibiotics and pathogenesis-related proteins targeted against secondary infection, which can account for enhanced resistance against bacterial and fungal pathogens but are not thought to control viral infection. This study showed that enhanced resistance against cucumber mosaic virus is caused by a tobacco calmodulin-like protein, rgs-CaM, which detects and counteracts the major viral virulence factor (RNA silencing suppressor) after SAR induction. rgs-CaM-mediated SAR illustrates the growth versus defense trade-off in plants, as it targets the major virulence factor only under specific biotic stress conditions, thus avoiding the cost of constitutive activation while reducing the damage from virus infection.

Useful ultrasonographic evaluation of the medial meniscus as a feature predicting the onset of radiographic knee osteoarthritis.

OBJECTIVE: Medial radial displacement (MRD) of the medial meniscus is a feature proving a dysfunction in the medial meniscus in osteoarthritis (OA) of the knee. MRD was measured in radiographic pre-OA knee and early osteoarthritis of the knee (early-OA) longitudinally using ultrasound (US) to investigate the characteristics involved in the onset and progression of OA. METHODS: Fifty-five patients with pain on the medial side of the knee participated in the present study. It was possible to follow-up 46 patients for 5 years, and, thus, they were divided into 32 pre-OA patients (female: 59%, mean age: 69.0 years) and 14 early-OA patients (female: 78%, mean age: 74.4 years) based on radiography at the baseline time-point. MRD was measured in standing and supine positions at baseline and after 1 and 5 years using US. MRD corrected with the skeletal size, i.e., the medial displacement index (MDI), was analyzed. The pre- and early-OA groups were divided into subgroups at 5 years: stable and OA progression groups, following the Kellgren/Lawrence classification, and ⊿MDI (gap of the MDI between the standing and supine positions) were retrospectively compared between the subgroups at baseline, 1 and 5 years. RESULTS: In the overall pre-OA group, MDI increased by 7% and 10% at 5 years in the supine and standing position, showing a significant increase (P = 0.044, 0.0147). ⊿MDI was significantly greater in the subgroup with OA progression in the pre- and early-OA groups (P = 0.02 and 0.03, respectively), and was continuously 6-7% in the pre-OA progression group, showing that the displacement rate was 2-fold or higher than in the stable group. CONCLUSION: An increase in ⊿MDI on US may be an important risk factor for the disease stage progression of OA and useful as a feature predicting the onset of radiographic knee OA.