Medicinal plant extracts protect epithelial cells from infection and DNA damage caused by colibactin-producing Escherichia coli, and inhibit the growth of bacteria.

AIMS: To investigate the biological activity of Thai medicinal plant extracts and their active substances on the inhibition of growth and the transcription of colibactin genes of colibactin-producing Escherichia coli, and effect on the pathogenesis from colibactin toxin including transient infections and colibactin-induced DNA damage. METHODS AND RESULTS: Among 16 medicinal plants examined, aqueous extracts of Terminalia catappa, Psidium guajava and Sandoricum koetjape demonstrated the growth inhibition against E. coli ATCC 25922, which is known to produce colibactin toxin. These plant extracts contain the active phytochemical compounds, tannin and quercetin, which are able to inhibit the growth of E. coli ATCC 25922. Interestingly, the extracts of T. catappa, P. guajava and S. koetjape, and their compounds tannin and quercetin, protected the eukaryotic epithelial cells of Vero cells and Caco-2 cells from infection and DNA damage by E. coli ATCC 25922. Moreover, these plant extracts and compounds exhibited efficacy to downregulate the expression of five genes (clbA, clbB, clbM, clbN and clbP) that are required for colibactin biosynthesis. CONCLUSIONS: The extracts of T. catappa, P. guajava and S. koetjape, and their compounds of tannin and quercetin had ability to inhibit the growth and transcription of colibactin genes of colibactin-producing Escherichia coli. Hence, these plant extracts and compounds could protect the transient infection and DNA damage of the eukaryotic epithelial cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first of its kind to report on the enhancement of the biological properties of T. catappa, P. guajava and S. koetjape, and to support the exogenous compound usage of tannin and quercetin, which may be able to protect against the transient infection and DNA damage of eukaryotic cells from E. coli carrying colibactin toxin.

A long-term follow-up study of open reduction using Ludloff's approach for congenital or developmental dislocation of the hip.

OBJECTIVES: There are several reports clarifying successful results following open reduction using Ludloff's medial approach for congenital (CDH) or developmental dislocation of the hip (DDH). This study aimed to reveal the long-term post-operative course until the period of hip-joint maturity after the conventional surgical treatments. METHODS: A long-term follow-up beyond the age of hip-joint maturity was performed for 115 hips in 103 patients who underwent open reduction using Ludloff's medial approach in our hospital. The mean age at surgery was 8.5 months (2 to 26) and the mean follow-up was 20.3 years (15 to 28). The radiological condition at full growth of the hip joint was evaluated by Severin's classification. RESULTS: All 115 hips successfully attained reduction after surgery; however, 74 hips (64.3%) required corrective surgery at a mean age of 2.6 years (one to six). According to Severin's classification, 69 hips (60.0%) were classified as group I or II, which were considered to represent acceptable results. A total of 39 hips (33.9%) were group III and the remaining seven hips (6.1%) group IV. As to re-operation, 20 of 21 patients who underwent surgical reduction after 12 months of age required additional corrective surgeries during the growth period as the hip joint tended to subluxate gradually. CONCLUSION: Open reduction using Ludloff's medial approach accomplished successful joint reduction for persistent CDH or DDH, but this surgical treatment was only appropriate before the ambulating stage. Cite this article: Bone Joint Res 2014;3:1-6.

The modified glucose clearance test: a novel non-invasive method for differentiating non-erosive reflux disease and erosive oesophagitis.

BACKGROUND: Impaired salivary secretion has been reported to cause abnormal acid clearance from the oesophagus in gastro-oesophageal reflux disease (GERD). However, few studies have explained the differences between non-erosive reflux disease (NERD) and erosive oesophagitis (EO) with respect to salivary secretion. Aim To elucidate these differences, we measured salivary secretion by using the modified glucose clearance test (mGCT). METHODS: All subjects completed endoscopic examinations, the frequency scale for the symptoms of GERD questionnaire and the mGCT comprising a resting GCT (measured as RGC time) and a chewing-stimulated GCT (SGC time). RESULTS: Resting glucose clearance time was 18.5 min in control group and significantly longer in NERD and EO groups (28.5 and 39.0 min respectively). SGC time was 6.1 min in control group and 7.2 min in NERD group and significantly longer in EO group (10.2 min) than in the control and NERD groups. CONCLUSIONS: In the EO group, both resting and stimulated salivary secretions were less than in control group. However, in the NERD group, resting salivary secretion was decreased, but stimulated salivary secretion was similar to that of the control group. Therefore, these results may help in explaining the differences in the pathogenesis of NERD and EO.

Symptoms of depression, prescription of benzodiazepines, and the risk of death in hemodialysis patients in Japan.

Many hemodialysis patients in Japan have symptoms of depression, but whether those patients are treated appropriately is unknown. As part of the Dialysis Outcomes and Practice Patterns Study, data on symptoms of depression, physician-diagnosed depression, prescribed medications, and death were collected prospectively in cohorts in Japan (n=1603) and 11 other countries (n=5872). Symptoms of depression were as prevalent in Japan as elsewhere, but in Japan a much smaller percentage of patients had physician-diagnosed depression: only 2% in Japan vs 17% elsewhere. Antidepressants were much less commonly prescribed in Japan: only 1% in Japan vs 17% elsewhere for patients with many and frequent symptoms of depression, and 16% in Japan vs 34% elsewhere for patients with physician-diagnosed depression. In Japan, symptoms of depression were associated with prescription of benzodiazepines (without antidepressants), and patients with physician-diagnosed depression were twice as likely to be given benzodiazepines: 32% in Japan vs 16% elsewhere. Benzodiazepine monotherapy was associated with death (relative risk 1.56, 95% confidence interval (CI), 1.25-1.94), even after adjustments for 13 likely confounders (relative risk 1.27, 95% CI, 1.01-1.59). Hemodialysis patients in Japan with symptoms of depression are given not antidepressants but benzodiazepines, a practice associated with higher mortality.

Disease-specific expression of VEGF and its receptors in AML cells: possible autocrine pathway of VEGF/type1 receptor of VEGF in t(15;17) AML and VEGF/type2 receptor of VEGF in t(8;21) AML.

Various angiogenic factors, such as vascular endothelial growth factor (VEGF) and an associated molecule, placenta growth factor (PlGF), are thought to be important for normal and malignant hematopoiesis. This study examined mRNA expression of VEGF, PlGF and receptors for these molecules in AML cells and identified the disease-specific patterns of expression. AML M3 having t(15;17) abnormality showed highest expression of VEGF and VEGF receptor type 1 (VEGFR1), suggesting the autocrine pathway of VEGF-VEGFR1. Then, t(8;21) AML demonstrated augmented expression of VEGF and VEGF receptor type 2 (VEGFR2), suggesting VEGF-VEGFR2 autocrine pathway. Then, addition of VEGFR2 kinase inhibitor in Kasumi-1, a t(8;21) AML cell line, resulted in marked inhibition of cell growth, although growth inhibitory effect of R2 kinase inhibitor to HL-60 was marginal. In addition, cell cycle analysis study showed S-phase cell population reduction by R2 kinase inhibitor in Kasumi-1, but not in HL-60. This observation is thought to be the rationale for novel molecular target therapy directed to angiogenic molecules.

Bacterial cysteine desulfurases: their function and mechanisms.

Cysteine desulfurase is a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme that catalyzes the conversion of L-cysteine to L-alanine and sulfane sulfur via the formation of a protein-bound cysteine persulfide intermediate on a conserved cysteine residue. Increased evidence for the functions of cysteine desulfurases has revealed their important roles in the biosyntheses of Fe-S clusters, thiamine, thionucleosides in tRNA, biotin, lipoic acid, molybdopterin, and NAD. The enzymes are also proposed to be involved in cellular iron homeostasis and in the biosynthesis of selenoproteins. The mechanisms for sulfur mobilization mediated by cysteine desulfurases are as yet unknown, but enzymes capable of providing a variety of biosynthetic pathways for sulfur/selenium-containing biomolecules are probably applicable to the production of cofactors and the bioconversion of useful compounds.

Cutaneous sarcoidosis successfully treated with topical tacrolimus.

Sarcoidosis is a disorder characterized by macrophage- and T-cell-mediated responses to as yet unidentified infectious antigens or autoantigens. We describe a 62-year-old woman with a 10-year history of orange-yellow plaques of sarcoidosis on her face. Her cutaneous lesions responded to topical tacrolimus ointment after unsuccessful treatment with topical and systemic corticosteroids. No adverse effects were noted with topical tacrolimus in this patient. We discuss the mode of action by which this immunosuppressive agent may act against sarcoidosis.

Up-regulation of the urokinase-type plasminogen activator receptor by monocyte chemotactic proteins.

The urokinase-type plasminogen activator receptor (uPAR) is a multifunctional molecule involved in migration and adhesion of leukocytes to sites of inflammation. Based on our hypothesis that a chemoattractant can stimulate uPAR expression by its target cell, thereby promoting cell migration, we employed three chemokines [monocyte chemotactic protein (MCP)-1, MCP-2 and MCP-3] as chemoattractants, and examined their effect on uPAR expression in a human monocyte-like cell line, U937. Northern blot analysis demonstrated that all three chemokines tested increased the level of uPAR mRNA in time-dependent and dose-dependent manners. Among them, MCP-3 exhibited the most potent effect. Scatchard analysis showed that incubation with MCP-3 (1 x 10(-8) mol/l) for 16 h resulted in a significant increase in the number of uPAR from (6.8 +/- 0.3) x 10(3) to (10.3 +/- 1.6) x 10(3)/cell, and in a slight increase in the equilibrium dissociation constant, K(d). The effect of anti-uPAR antibodies on MCP-3-induced U937 cell migration across an endothelial cell monolayer and a type I collagen layer was assessed by means of the modified Boyden chamber assay. Although MCP-3 caused a three-fold increase in migration, incubation with an antibody to uPAR markedly abrogated the induced cell migration. These results support our hypothesis and suggest that up-regulation of uPAR in target cells might be an important and common feature of chemoattractants.

Expression of endothelial cell-associated molecules in AML cells.

Recently, it has been clarified that interaction between hematopoietic cells and endothelial cells is important in normal hematopoiesis and leukemogenesis. In this study, we examined the relationship between AML cells and endothelial cells by analyzing the expression profile of angiogenic factors, angiopoietin-1 (Ang-1), Ang-2, Tie-2 (a receptor for angiopoietins) and vascular endothelial growth factor (VEGF). Our results demonstrated that CD7(+)AML expressed Ang-2 mRNA frequently and integrin-family adhesion molecules (CD11c and CD18) intensively, suggesting the close correlation with endothelial cells. On the other hand, in t(8;21) AML cells, expression of Ang-2 was infrequent and expression of integrin-family adhesion molecules (CD11b, CD11c and CD18) was weak, suggesting the sparse association with endothelial cells. As for CD7(+)AML cells, despite the frequent and intense expression of endothelial cell-associated molecules (such as Ang-2, CD11c and CD18), intensity of Tie-2 expression was quite low (P < 0.05). Ang-2 expressed in CD7(+)AML cells is not considered to act in an autocrine fashion, but to work on endothelial cells to "feed" leukemic cells. Although Ang-2 is recognized as a natural antagonist for Tie-2, our data presented here suggested the alternative role of Ang-2 in the relationship between endothelial cells and leukemia cells, at least in a subset of leukemia such as CD7(+)AML. These results were supported by the study using AML cell lines, KG-1 (CD7 negative) and its subline KG-1a (CD7 positive); KG-1 had mRNA expression profile of Ang-1(+)Ang-2(-)Tie-2(+), while KG-1a showed Ang-1(+)Ang-2(+)Tie-2(-). These difference in the expression profile of angiogenic factors between CD7(+)AML and t(8;21)AML may explain the characteristic morphological features of these leukemias (CD7(+)AML as blastic type and t(8;21)AML as differentiative type).

Follow-up study of conservative treatment for atlantoaxial rotatory displacement.

Rotatory displacement of the atlantoaxial joint is one of the causes of torticollis in children. Some of these cases show persistent symptoms and may lead to rotatory fixation; however, only a few studies have been directed to the prognosis of atlantoaxial rotatory displacement after conservative treatment. Clinical and radiographic reviews were performed in 35 patients (16 male and 19 female) with rotatory displacement of the atlantoaxial joint. The mean age at admission was 6.5 (range: 2-12) years old, and the mean follow-up period was 3.4 (1.4-5.8) years. All patients successfully achieved reduction after 2 to 3 weeks of continuous halter traction; however, 9 patients (25.7%) experienced recurrence, and 7 patients (20.0%) had a slight torticollis at follow-up. The duration of symptoms before treatment affected the recurrence rate, and the torticollis was apt to remain in cases with severe displacement at admission.

Cervical posterior fusion with wave-shaped rod under local anesthesia for cervical spondylotic myelopathy: review of 12 patients.

STUDY DESIGN: Clinical evaluation of cervical interspinous fusion under local anesthesia in elderly patients with cervical spondylotic myelopathy. OBJECTIVES: To evaluate the effectiveness of cervical posterior fusion with wave-shaped rods inserted under local anesthesia for elderly high-risk patients with cervical spondylotic myelopathy. SUMMARY OF BACKGROUND DATA: A substantial number of patients cannot undergo surgical interventions under general anesthesia because of their general medical complications. Although such patients would become unable to walk, which might induce a worsening of their general condition, conservative treatments had been adopted as the only treatment for these patients. The authors have obtained satisfactory results by means of posterior interspinous fusion under local anesthesia even in the high-risk patients with severe cervical spondylotic myelopathy. The aims of this surgical technique were to adjust cervical alignment and to stabilize the motion segment(s) without decompression. PATIENTS AND METHODS: Between May 1989 and August 1998, 12 elderly patients (3 men and 9 women) with cervical spondylotic myelopathy were treated with posterior interspinous fusion using wave-shaped rods inserted under local anesthesia. The average age at the surgery was 76.9 years. The average follow-up period was 5 years 6 months. All patients were unable to walk without any assistance because of their advanced myelopathy. It was felt that all of them would be unable to accept general anesthesia because of their generally poor medical conditions. Preoperative severity of the clinical symptoms and postoperative recovery were evaluated by a scoring system proposed by the Japanese Orthopaedic Association, which had 17 points at full mark. RESULTS: The average duration of the surgical procedure was 122.8 minutes. The average total blood loss was 118.6 g. No instrument failures were denoted. Neither neural deterioration nor major complication was observed relating to the surgery. Radiographic bony union of the grafted bone was achieved in all patients. Progression of myelopathy was arrested in all 12 patients, and clinical symptoms were improved in 10 patients. The mean Japanese Orthopaedic Association scores had increased from 5.0 to 10.2 points. CONCLUSIONS: Twelve high-risk patients with cervical spondylotic myelopathy were treated with posterior interspinous fusion using wave-shaped rods inserted under local anesthesia. This method was evaluated as an effective surgical salvage without any mortal complications even in the elderly high-risk patients.

Removal of soluble selenium by a selenate-reducing bacterium Bacillus sp. SF-1.

In order to develop a biological process for removal of selenium from industrial wastewater, Bacillus sp. strain SF-1 was isolated from selenium-contaminated sediment. The bacterium reduces selenate to selenite and subsequently to nontoxic insoluble elemental selenium using lactate as an electron donor and selenate as an electron acceptor in an anaerobic condition. Elemental selenium transformed from soluble selenium was deposited both inside and outside of the cells. Since the selenate reduction rate of the strain SF-1 was higher than the selenite reduction rate, selenite was transiently accumulated. In an experiment of the repeated soluble selenium reduction by strain SF-1, 0.5 mM of selenate was sequentially treatable with a cycle of one day. Thus, our sequential system for removal of soluble selenium is very useful.

Multipeptide-metalloporphyrin assembly on a dendrimer template and photoinduced electron transfer based on the dendrimer structure.

To construct an artificial photosynthetic system, peptide dendrimers [n-(X-HLY)PAMAMs: X = R, E; Y= L, F; n=4, 8, 16, 32 and 64 segments], in which amphiphilic alpha-helix peptides (X-HLY: R-HLL, E-HLL and R-HLF) were introduced at the end groups of polyamidoamine dendrimers (PAMAMs), were designed and synthesized. The peptide dendrimers 64-(X-HLY)PAMAMs are novel synthetic biopolymers with an enormous molecular weight, about 160 kDa, and with a regulated amino acid sequence and three-dimensional conformation. The peptide dendrimers bound Fe(III)- or Zn(II)-mesoporphyrin IX per two alpha-helices; this afforded a multimetalloporphyrin assembly similar to the natural light-harvesting antennae in photosynthetic bacteria. Circular dichroism studies and peroxidase activity measurements revealed that metalloporphyrins were coordinated to the peptide dendrimers in a regulated manner and packed more densely with the growth of the dendrimer generation. Fluorescence quenching and photoreduction studies with methylviologen demonstrated that the photoinduced electron-transfer function with the peptide dendrimer-multi-Zn-MP was accomplished more effectively as the dendrimer generation increased. Thus, the three-dimensional assembly of metalloporphyrins and peptides in the dendrimer was an effective module for light-harvesting antennae in an artificial photosynthetic system.

Biomechanical comparison of posterior cervical fixation.

STUDY DESIGN: Biomechanical stability using four different posterior cervical fixation techniques was evaluated in human cadaveric spine. OBJECTIVES: To introduce an alternative interspinous fixation technique using wavy-shaped rods, and to compare its in vitro biomechanical stability with that of other posterior cervical fixation techniques. SUMMARY OF BACKGROUND DATA: Fixation of the posterior cervical spine with interspinous wiring is well known as Rogers' or Bohlman's technique. Recently, several plate fixation techniques have been used for posterior cervical stabilization. Since 1983, the authors have developed the wavy-shaped rod system as an alternative to the interspinous fixation technique. This unique technique has been proven clinically useful in Japan. However, the authors are not aware of any prior biomechanical studies. METHODS: Seven fresh frozen cervical human spines were tested at the C5-C6 motion segment. Nondestructive static biomechanical testing was performed with flexion-extension, lateral bending, and axial rotation for the following stabilization techniques: intact spine, creation of a Stage 3 distractive-flexion injury followed by fixation with the wavy-shaped rods bounded by three multistrand cables, interspinous wiring with a multistrand cable, triple wiring technique using multistrand cables with a pair of unicortical grafts from the ilium, and lateral mass plate fixation with Magerl's screw technique. Testing was performed on a material testing machine (MTS 858 Bionix test system, MTS, Minneapolis, MN), and load displacement curves were obtained using four linear extensometers and one rotatory extensometer across the C5-C6 motion segment. RESULTS: In axial compression loading, the reconstructed specimens showed significant differences in range of motion measured at the anterior and posterior positions, and statistical analysis was performed using one-way analysis of variance. In a comparison of the four fixation techniques, the construct with the wavy-shaped rod indicated significantly less motion both anteriorly and posteriorly than with the other fixation techniques. Also in flexion-extension loading, all the techniques significantly limited the intervertebral motion below the level of the intact motion segment. Particularly, the construct with the wavy-shaped rod showed the smallest mobility, 49.9% anteriorly and 9.3% posteriorly, compared with that of the intact spine. In lateral bending, the lateral mass plate provided the greatest stability, which was superior to the intact segment, but the difference was not statistically significant. In axial rotation, all the reconstruction techniques limited the angular motion below the intact level (wavy rod, 68.0%; Rogers' wiring, 75.2%; Bohlman's triple wiring, 59.8%; lateral mass plate, 71.7%), but no significant differences were observed using one-way analysis of variance, as compared with the intact segment. CONCLUSIONS: All four reconstruction techniques restored the stability of the cervical motion segment to at least the level of the intact motion segment before destabilization. An alternative cervical posterior fixation technique, the Wavy Rod system, was considered the most effective technique in stabilizing a cervical motion segment, particularly in axial compression and flexion extension loading.

Malignant lymphoma of the bone associated with systemic sarcoidosis.

A 57-year-old woman was hospitalized with malignant lymphoma of the right talus. After treatment, complete remission was obtained. Gallium-67 scintigraphy to confirm the remission demonstrated increased uptake in the whole body skeletal muscle, especially in her thighs. Biopsy of right gastrocnemius muscle showed epithelioid granuloma. Serum angiotensin-converting enzyme activity (ACE) and lysozyme had increased to several times the normal range. We diagnosed her disease as bone-associated sarcoidosis-lymphoma syndrome. Human herpes virus 8 (HHV-8) genome was examined in the bone marrow specimen, and the relationship between sarcoidosis-lymphoma syndrome and HHV-8 was discussed.

Construction of peptide conjugates with peptide nucleic acids containing an anthracene probe and their interactions with DNA.

We designed and synthesized the peptide nucleic acid (PNA)-peptide conjugates having anthracene chromophores and investigated their interactions with calf thymus DNA, [d(AT)(10)](2), [d(GC)(10)](2), and [d(AT)(10)dA(6)](2). Considering the synthesis compatibility and expecting that a novel DNA analogue, PNA, can improve DNA binding properties of alpha-helix peptides, we attempted to attach thymine PNA oligomers at the C-terminus of a 14 amino acid alpha-helix peptide that contained a pair of artificial intercalators, anthracene, as a probe, and to examine their interactions with DNA using anthracene UV, fluorescence and circular dichroism properties. The results observed in this study showed that the designed peptide folded in an alpha-helix structure in the presence of calf thymus DNA, [d(AT)(10)](2), and [d(AT)(10)dA(6)](2) with the chromophores at the side-chain being fixed with a left-handed chiral-sense orientation. The alpha-helix and the anthracene signals were not observed for [d(GC)(10)](2). Incorporation of thymine PNA oligomers into the designed alpha-helix peptide increased the DNA binding ability to [d(AT)(10)dA(6)](2) with increasing the length of the PNA without changing the conformations of the peptide backbone and the anthracene side-chains.

Design and synthesis of 3alpha-helix peptides forming a cavity for a fluorescent ligand.

As a model of receptor protein, a series of 3alpha-helix bundle peptides constructed on a template peptide were designed so as to possess a hydrophobic cavity. The size of cavity was modulated by simple replacements of Leu residues to Ala residues in the hydrophobic core. Binding abilities to 8-anilino-1-naphthalenesulfonic acid (ANS) were estimated by the increase of fluorescence intensity. The peptide having three or four Ala residues in the hydrophobic core remarkably increased the binding ability for ANS, though the peptide having two Ala residues gave an inefficient cavity for ANS. The peptide having six Ala residues decreased the binding ability due to crucial destabilization of the helix bundle structure. This scaffold can be utilized to a receptor model, while further tuning of the sequence is necessary.

Photoinduced hydrogen evolution with peptide dendrimer-multi-Zn(II)-porphyrin, viologen, and hydrogenase.

To construct an artificial photosynthetic system, multi-Zn(II)-mesoporphyrins in peptide dendrimers were equipped as a photosensitizer of photoinduced hydrogen evolution in a four-component system (electron donor, photosensitizer, electron carrier, and catalyst), so that hydrogen was evolved effectively by the dendrimer architecture, for the first time. The hydrogen evolution activity was correlated to the photoreduction ability of viologen by the Zn-porphyrin-peptide dendrimers. Additionally, using positively charged methyl-viologen as an electron carrier, the photoinduced hydrogen evolution function with the positively charged peptide dendrimer was superior to that with the negatively charged peptide dendrimer, despite that the positive dendrimer did not strongly bind the positively charged methyl-viologen with the electrostatic interaction. By contrast, when zwitterionic propylviologen sulfonate was used, photoreduction and hydrogen evolution properties were identical between the positively and the negatively charged dendrimers. These results demonstrated that the dynamic interaction between the positive dendrimer and methyl-viologen was preferable for the photoreduction and hydrogen evolution, and that the three-dimensional assembly of Zn(II)-mesoporphyrins using the peptide dendrimers was effective as a photosensitizer in the artificial photosynthesis.